CN113122568A - Method for improving corn biomass - Google Patents

Abstract

The invention discloses a method for improving corn biomass, which comprises the following steps: inserting a regulatory sequence into the upstream or downstream of a target gene of a corn genome at a fixed point, and screening to obtain corn with improved biological quantity; the target gene is PLA1 gene or TE1 gene, and the regulatory sequence is shown in SEQ ID NO. 3. Compared with the parental control, the improved corn greatly improves the whole plant dry weight, the dry ear weight and the hundred grain weight, particularly improves the single plant ear yield by more than 25 percent, and has application prospect in breeding new corn varieties.

Description

Method for improving corn biomass

(I) technical field

The invention relates to a technology for improving the yield and characters of corn by using a gene editing technology, belonging to the technical field of biology.

(II) background of the invention

Corn is a major food crop and also a good feed and industrial raw material. The corn has the characteristics of high heat, high straw yield, rich vitamins in the straw and the like. The corn planting cost is relatively low, and with the rapid development of animal husbandry, the demand of corn as feed is gradually increased. In recent years, the corn whole plant silage mode is popularized in various places. The corn used as silage not only pursues the corn kernel yield, but also pays attention to the yield of the non-kernel part of the corn, and the value of improving the overall biological yield of the corn in production application is higher and higher.

The existing research finds that the maize leaf primordium formation interval is a key factor influencing the growth and development of maize, and the key genes Plastochron1(PLA1) and Terminal ear1(TE1) influencing the leaf primordium formation interval are identified. Studies have shown that deletion of TE1 expression results in maize internode shrinkage, maize plants that are dwarf and leaf-diminished (Veit, Nature,1998,393: 166-168). The over-expression of the TE1 gene can obviously increase the biological yield and the seed number, and improve the harvest index and the grain weight (Zhang Xian, 2017100464476). Overexpression of PLA1 in maize can significantly improve maize yield while also enhancing maize stress tolerance (Sun, Nature communications,2017, 8: 14752; Shenshicheng, 2015102305475). High expression of PLA1 and TE1 genes in maize contributes to increased biological yield in maize. The current general method is to introduce an artificial expression frame for enhancing PLA1 or TE1 gene expression and T-DNA containing a selection marker gene into a plant by using a transgenic method to improve the expression level of the PLA1 or TE1 gene in corn. The use of transgenic approaches also typically requires the use of a selection marker gene that is tightly linked to the enhanced expression cassette of PLA1 or TE1 and cannot be isolated later by breeding programs. The introduction of the selection marker gene into corn may increase the ecological risk to the environment in the cultivation and production of corn and the safety risk in the edible feed. In addition, random insertion of expression cassettes containing PLA1 or TE1 into the maize genome may also affect the expression of genes around the insertion site, and thus may have unpredictable effects on maize traits.

The invention utilizes gene editing technology to introduce regulatory sequences into a corn genome at fixed points to improve the expression level of PLA1 or TE1 genes in corn, thereby obtaining corn with improved biological yield and enhanced growth potential. By backcross transformation, screening marker genes used in the process of gene editing and screening can be effectively removed, the breeding efficiency of new corn varieties is improved, and the planting and application risks of genetically modified corns are reduced.

Disclosure of the invention

The invention aims to provide a method for improving corn yield, and particularly relates to a method for improving the expression of a key gene PLA1 or TE1 gene in a corn leaf primordial phase by using a gene editing method, so that the corn biological yield is improved.

The technical scheme adopted by the invention is as follows:

the invention provides a method for improving corn biomass, which introduces a regulatory sequence to the upstream or downstream of a target gene in a corn genome to enhance the expression of the target gene, thereby improving the growth potential of corn and improving the agronomic traits of corn, and the specific method comprises the following steps: inserting a regulatory sequence into the upstream or downstream of a target gene of a corn genome at a fixed point to obtain corn with improved biological quantity; the target gene is PLA1 gene or TE1 gene, and the nucleotide of the regulatory sequence is shown in SEQ ID NO. 3.

Further, the PLA1 gene is a key gene of a leaf primordium formation interval period, and the nucleotide sequence of the PLA1 gene is shown in SEQ ID NO. 1; the upstream insertion site of the PLA1 gene is between the 247306010 and 247312010 bases of the No.1 chromosome of maize; the downstream insertion site of the PLA1 gene is between the bases of the maize No.1 chromosome 247314293 and 247317293.

Further, the TE1 gene is a key gene of a leaf primordium formation interval period, and the nucleotide sequence of the TE1 gene is shown in SEQ ID NO. 2; the upstream insertion site of the TE1 gene is between 167811126 and 167821126 bases of the maize chromosome 3; the downstream insertion site of the TE1 gene is between 167824995 and 167834995 bases of the maize chromosome 3.

Designing a gene editing vector containing a marker gene according to an insertion site, synthesizing a recombinant donor fragment containing a regulatory sequence, introducing the gene editing vector containing the marker gene and the recombinant donor fragment containing the regulatory sequence into young maize embryos by using a gene gun method, removing transgenic maize containing the marker gene, and screening to obtain the gene editing maize with improved biological quantity; the recombination donor sequence is formed by sequentially connecting an upstream homology arm of a target gene insertion site, a regulatory sequence and a downstream homology arm of the insertion site; the vector containing the marker gene comprises a Cas9 gene expression cassette, an upstream sgRNA coding sequence and a downstream sgRNA coding sequence.

The gene editing vector containing the marker gene takes pCambia1300(GenBank: AF234296.1) as a Plant transformation vector framework, takes a glyphosate-resistant gene expression frame as the marker gene, synthesizes a Cas expression frame and a target recognition expression frame according to the method described by Yongwei Sun et al (Molecular Plant,2016,9:628-631), constructs a gene editing vector P-G10-sgRNA-Cas 9-sgRNAB, and has the full sequence shown as SEQ ID NO. 4.

When the insertion site of the enhanced sequence is at the upstream of a PLA1 expression frame of a target gene, the insertion site is between 247306010-247312010 bases of a No.1 chromosome of corn (gene locus information is based on a whole genome sequence of B73 in a corn genomic database website www.maizegdb.org); designing an upstream homology arm (SEQ ID NO.5) and a downstream homology arm (SEQ ID NO.6) of the enhanced sequence according to the insertion site; sequentially connecting an upstream homologous arm of an insertion site shown in SEQ ID NO.5, a regulatory sequence shown in SEQ ID NO.3 and a downstream homologous arm of an insertion site shown in SEQ ID NO.6 to form a recombinant donor sequence; a sgRNA target is designed at 247310934 bases of a No.1 chromosome of corn, and is named as sgRNA1, and the sequence is as follows: acctccgcctcgcccgaccc, designing another target at 247310995bp, named sgRNA2 with sequence catcgcccaacctagggcgc; replacing a sgRNA sequence in a gene editing vector P-G10-sgRNA-cas 9-sgRNA shown in SEQ ID NO.4 with sgRNA1, and replacing the sgRNA sequence with sgRNA2 to obtain a PLA1 upstream gene editing vector P-G10-sgRNA1-cas9-sgRNA 2; adopting a gene gun bombardment method, and carrying out gene editing on a vector and a corresponding recombinant donor fragment according to the ratio of 1: 20 mol ratio, introducing into young embryo of corn, screening to obtain transgenic corn with inserted enhancement sequence, and separating and screening marker gene to obtain gene-edited corn.

When the insertion site of the enhancement sequence is downstream of the expression frame of the PLA1 gene, the site-specific insertion site is between the bases of the maize No.1 chromosome 247314293 and 247317293; designing a sgRNA target at the 247314496bp site of the No.1 chromosome of the corn, and naming the sgRNA target as 3 and gttcactaaagaacttaatc; 247315566bp sites are used for designing a target, namely sgRNA4, atcaattcaagcacttaaac; designing an upstream homology arm according to the insertion site, wherein the nucleotide sequence is SEQ ID NO.7, the downstream homology arm is SEQ ID NO. 8; the insertion method is the same as the insertion of the enhancement sequence upstream of the PLA1 gene expression cassette.

When the site-specific insertion site provided by the invention is upstream of a TE1 gene expression frame, the insertion site of a regulatory sequence is between 167811126 and 167821126 bases of a No.3 chromosome of corn, and the genetic locus information is according to a whole genome sequence of B73 in a corn genomic database website www.maizegdb.org; a sgRNA target is designed at 167822232bp of a maize 3 # chromosome, is named as sgRNA5, and has the sequence: aggtgttgtctaggcttggc, designing another target at 167812349bp, named sgRNA6 with sequence gtcggacgatccgcgcctat; designing an upstream homology arm according to the insertion site, wherein the nucleotide sequence is SEQ ID NO.9, and the nucleotide sequence of a downstream homology arm is SEQ ID NO. 10; the insertion method is the same as the insertion of the enhancement sequence upstream of the PLA1 gene expression cassette.

When the fixed point insertion site provided by the invention is downstream of a TE1 gene expression frame, the enhancement sequence insertion site is between 167824995-167834995 bases of a No.3 chromosome of maize; a sgRNA target is designed at 167825705bp of a maize 3 # chromosome, is named as sgRNA7, and has the sequence: ttcttataaataaatatttt, designing another target at 167825814bp, named sgRNA8 with sequence cttgaagtgagatcttaaat; designing an upstream homology arm according to the insertion site, wherein the nucleotide sequence is SEQ ID NO.11, the downstream homology arm is SEQ ID NO. 12; the insertion method is the same as the insertion of the enhancement sequence upstream of the PLA1 gene expression cassette.

Compared with the prior art, the invention has the following beneficial effects:

compared with the parental control, the improved corn greatly improves the whole plant dry weight, the dry ear weight and the hundred grain weight, particularly improves the single plant ear yield by more than 25 percent, and has application prospect in breeding new corn varieties.

(IV) description of the drawings

FIG. 1 is a diagram of a carrier frame.

FIG. 2 is a diagram of a recombinant donor fragment structure.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: preparation of Gene editing plasmids and Donor sequences

(1) Target design

The nucleotide sequence of the PLA1 gene is shown in SEQ ID NO. 1.

When the enhancement sequence is inserted into the upstream of the PLA1 gene expression cassette, the site-specific insertion site is between 247306010 and 247312010 bases of the No.1 corn chromosome, and the gene site information is based on the whole genome sequence of B73 in the corn genomic database website www.maizegdb.org. One sgRNA target was designed at 247310934 base of chromosome 1 in maize, designated sgRNA1, and the other target was designed at 247310995bp, designated sgRNA 2.

When the enhancement sequence is inserted into the downstream of the PLA1 gene expression frame, the site-specific insertion site is between the bases of the No.1 chromosome 247314293 and 247317293 of the maize; designing an sgRNA target in an 247314496bp design insertion segment of a maize chromosome 1, and naming the sgRNA 3; an 247315566bp site designed a target, designated sgRNA 4.

When the enhanced sequence is inserted into the upstream of the TE1 gene expression cassette, the site-specific insertion site is between 167811126 and 167821126 bases of the maize chromosome 3, and the gene site information is according to the whole genome sequence of B73 in the maize genomic database website www.maizegdb.org. One sgRNA target was designed at 167822232bp of maize chromosome 3 and designated sgRNA5, and the other target was designed at 167812349bp and designated sgRNA 6.

When the enhancement sequence is inserted into the downstream of the TE1 gene expression cassette, the site-specific insertion site is between 167824995-167834995 bases of the No.3 chromosome of maize; one sgRNA target was designed at 167825705bp of maize chromosome 3 and designated sgRNA7, and the other target was designed at 167825814bp and designated sgRNA 8. The sequences of the respective target sites are shown in Table 1.

TABLE 1 target sites and sequences

(2) Gene editing vector

pCambia1300(GenBank: AF234296.1) is used as a Plant transformation vector framework, Shanghai biology, Inc. is entrusted to synthesize a marker gene glyphosate-resistant gene expression frame, a Cas expression frame and a target recognition expression frame are synthesized according to a method described by Yongwei Sun et al (Molecular Plant,2016,9:628-631), a gene editing vector P-G10-sgRNAa-Cas9-sgRNAB is constructed, the whole sequence is shown as SEQ ID NO.4, and the vector structure schematic diagram is shown as FIG. 1. The 1 st to 3740 th basic groups in SEQ ID NO.4 are glyphosate-resistant gene expression frames which are used as screening marker genes of transgenic corn; the 3741-position 3832 base is a T-DNA left arm sequence, the 3833-position 10065 base is a vector framework sequence, the 10066-position 10091 base is a T-DNA right arm sequence, the 10066-position 10836 base is a U3 promoter, the 10837-position 10856 base is a target recognition sequence, and the coded sgRNA is marked as sgRNA; bases 10857-17417 are Cas9 expression frame, gene editing enzyme; 17418 and 17923 is the U3 promoter; 17924 and 17943 base as target recognizing sequence, and encodes sgRNA, which is marked as sgRNA B, 17944 and 18041 base as CMV35S terminator sequence.

Replacing the sgRNA sequence in the gene editing vector P-G10-sgRNA-cas 9-sgRNA with sgRNA 1; replacing the sgRNA2 with the sgRNA sequence to obtain a PLA1 upstream gene editing vector P-G10-sgRNA1-cas9-sgRNA 2;

replacement of the sgRNA sequence to sgRNA 3; replacing the sgRNA4 with the sgRNA sequence to obtain a PLA1 downstream gene editing vector P-G10-sgRNA3-cas9-sgRNA 4;

replacement of the sgRNA sequence to sgRNA 5; replacing the sgRNA-B sequence with sgRNA6 to obtain TE1 upstream gene editing vector P-G10-sgRNA5-cas9-sgRNA 6;

replacement of the sgRNA sequence to sgRNA 7; the sgRNA8 was substituted with the sgRNA sequence to obtain the TE1 downstream gene editing vector P-G10-sgRNA7-cas9-sgRNA 8.

(3) Recombinant donor sequences

The nucleotide sequence of the regulatory sequence is shown as SEQ ID NO.3, the donor fragment is obtained by sequentially connecting an upstream homology arm, the regulatory sequence and a downstream homology arm, the structural schematic diagram is shown as figure 2, and the specific sequence structure is as follows:

PLA1 upstream recombination donor sequence H1: the sequence from the 5 'end to the 3' end is SEQ ID NO.5, SEQ ID NO.3, SEQ ID NO. 6;

PLA1 downstream recombination donor sequence H2: the sequence from the 5 'end to the 3' end is SEQ ID NO.7, SEQ ID NO.3, SEQ ID NO. 8;

TE1 upstream recombination donor sequence H3: the sequence from the 5 'end to the 3' end is SEQ ID NO.9, SEQ ID NO.3, SEQ ID NO. 10;

TE1 downstream recombination donor sequence H4: the sequence from 5 'end to 3' end is SEQ ID NO.11, SEQ ID NO.3, SEQ ID NO. 12.

Example 2: obtaining Gene-edited maize

The exogenous gene introduction is carried out by adopting a gene gun bombardment method, and the operation method, the steps and the culture medium formula are described by Joshi et al (J Microbiol Biotechnol Food Sci, 2016,5: 335-.

Selecting and pollinating 10-12 days, collecting well-grown corn inbred line B104 cluster, collecting young embryo (with size of 1.0-1.5mm), and culturing on induction culture medium for 3-4 days. Before gene gun bombardment, the immature embryo tissue is inoculated into the middle part of a hypertonic culture medium containing 36.4g/L mannitol and 36.4g/L sorbitol, the diameter of the medium is about 2.5 cm, and the medium is subjected to osmotic treatment for 4 hours.

Gene editing plasmids and corresponding recombinant donor fragments were expressed according to 1: 20 molar ratios were mixed to form four gene-editing DNA combinations as follows:

PLA1 upstream editing DNA combinations: P-G10-sgRNA1-cas9-sgRNA2+ H1;

PLA1 downstream editing DNA combinations: P-G10-sgRNA3-cas9-sgRNA4+ H2;

TE1 upstream editing DNA combinations: P-G10-sgRNA5-cas9-sgRNA6+ H3;

TE1 downstream editing DNA combinations: P-G10-sgRNA7-cas9-sgRNA8+ H4.

The DNA editing combination is respectively mixed with gold powder, the concentration of the gold powder is 1.0mmol/L, the bombardment pressure is 1100psi, the bombardment distance is 6 cm, each culture dish is bombarded for 1 time, the vacuum degree is adjusted to 24-27 inchwg, and each combination is respectively and independently implanted into a maize immature embryo.

After bombardment of young maize embryos, the embryos are transferred to an induction medium for dark culture for 16 hours and then transferred to a screening medium containing 2mM glyphosate. Screening for 2-3 rounds, selecting the resistant callus with good activity, transferring the resistant callus to a differentiation culture medium for differentiation, transferring the maize differentiated into seedlings to a rooting culture medium, culturing by illumination until the roots are completely developed, and transplanting the maize seedlings to a greenhouse for molecular detection to detect whether a regulatory sequence is inserted.

Example 3: identification of Gene-edited maize

Extracting maize genome DNA to be detected, taking the genome DNA as a template, and adopting a primer 1F (ACTCTCGTCTACTCCAAGAA) and a primer 1R (CAATCCATTGCTGAAGA) to carry out PCR amplification, wherein a parental control can not generate a band, and a specific band with the size of 300bp is generated by gene editing with the insertion of a regulatory gene. Screening to obtain Gene-edited maize lines containing regulatory Gene insertions is shown in Table 2

TABLE 2

Example 4: gene editing maize marker gene isolation

The maize obtained in example 3 was used as a male parent and a maize inbred line B104 was used as a female parent, and the female ear and the male flower were bagged, respectively, and after flowering, the maize pollen edited by the gene was pollinated to the B104 female ear, and after maturation, the maize was sun-dried and sown for screening of the gene-edited maize containing no selection marker gene. Detecting the corn progeny plants by using a G10 gene detection test paper (Youlong bio-company), removing positive plants containing G10 gene, carrying out PCR molecular detection on the rest non-transgenic corn, carrying out PCR amplification by using a primer 1F (ACTCTCGTCTACTCCAAGAA) and a primer 1R (CAATCCACTTTGCTTTGAAGA), generating a specific strip with the size of 300bp, and carrying out sequencing analysis on the strip. Screening to obtain the gene-edited corn without the G10 screening marker, and using the corn for later-stage variety transformation.

Example 5: gene editing maize biological yield comparison

The yield comparison test was performed on the gene-edited maize selected in example 4 without the G10 selection marker, against parental inbred line B104. The method adopts a completely random block design, the corn planting area is 20 square meters, 5 rows are arranged, the row length is 6 meters, the row spacing is 0.67 meter, the plant spacing is 0.22 meter, 27 holes are sowed in each row, 2-3 seedlings are sowed in each hole, one seedling is left in the process of final singling, and the yield is measured by sampling in the middle 3 rows after the corn is matured. The yield results show that the individual biomass yield, ear weight and ear grain weight of the gene-edited maize were significantly higher than the control maize.

TABLE 3

Note: indicates that the difference was extremely significant.

Sequence listing

<110> Hangzhou Ruifeng Biotechnology Ltd

<120> a method for increasing corn biomass

<160> 12

<170> SIPOSequenceListing 1.0

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<211> 1834

<212> DNA

<213> Unknown (Unknown)

<400> 1

atggcgatgg cctccgcggc ttgctcatgc acggacggca cgtggtgggt gtacgcgctc 60

ccggcgctgc tcggctccga caccctgtgc gcccacccgg ccctcctggc tggcctgatc 120

tttctggcca ccgtctcggt ggctctgctg gcgtgggcca cgtcgccggg cggtccggcg 180

tggacgaacg gccgcggccg cctcggcgtc actcctatcg tgggaccccg tggtctgccc 240

gtgttcggca gcatcttcgc gctgtcccgc gggctgccgc accgcgccct cgccgagatg 300

gcccgcgccg cagggccccg ggccaaggag ctcatggcgt tctccgtcgg tgacacgccc 360

gcggtcgtgt cgtcctgccc ggccacggca cgtgaggtgc tcgcgcaccc gtcattcgcc 420

gaccgccctg tgaagcggtc ggcccgggag ctcatgttcg cgcgtgccat cgggttcgcg 480

cccaacggcg agtactggcg ccgcctccgc cgcgtcgcgt ccacgcacct attctccccg 540

cgccgggtcg cctcgcacga gccgggacgc caaggtgacg cggaggccat gctccgctcc 600

atcgccgccg aacagtcggc ctctggcgcc gtcgccctcc gcccgcacct ccaggccgcc 660

gctctcaaca acatcatggg cagcgtcttc ggcacgcggt acgacgtcac atcaggcgcc 720

ggcgccgcgg aggccgagca tctcaagagc atggtgcgcg aggggttcga gctcctcggc 780

gccttcaact ggtccgacca cctcccctgg ctcgcccacc tgtacgaccc aagcaacgtc 840

acccgccggt gcgccgcgct cgtgccgcgc gtccagacct tcgtccgtgg cgtcatcgac 900

gagcaccggc gccgccgcca aaactccgcc gccctcaacg acaatgctga cttcgtcgac 960

gtgctcctct ccctcgaggg tgacgagaag ctcggcgacg acgacatggt cgccatcctc 1020

tgggtaaagt tcaaatcgat cgctttccta gcttgtttaa ctgcgcatac ttctcagttc 1080

tcaactgcgc atacctgtcg gttctacagt tttgtgtcgg gctgtcggtt gttcccggaa 1140

gggaaaaaaa agaacaaagc tctgtcgctg aaaaaaacat actgtacatg catataattt 1200

gtttttgcag gagatggtct tccgcggtac ggacacgacg gcgcttctga ccgagtggtg 1260

catggcggag ctggtgcgcc acccggcggt gcaggcgagg gtgcgcgccg aggtcgacgc 1320

ggctgtcggt gccggaggtt gccccaccga cgccgacgtg gcgcgcatgc cgtacctgca 1380

ggcggttgtg aaggagacgc tgcgcgccca cccgcctggc ccgctgctga gctgggctcg 1440

cctcgccacc gccgacgtgc cactctgcaa cggcatggtg gtcccggctg gcaccacggc 1500

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cgcgccggag cggttcctgc cctccgaggg cggcgccgac gtggacgtcc gcggcgtcga 1620

cctccgcctg gccccgttcg gcgccgggcg tcgcgtctgc cccggcaaga acctgggcct 1680

caccaccgtg ggcctctggg ttgcccgcct cgtgcacgcc ttccagtggg ccctgcctga 1740

cggcgcggcg gccgtttgcc tcgacgaggt cctcaagctc tccctggaga tgaagacgcc 1800

gctcgtcgcc gcagccatcc cccgcaccgc ctga 1834

<210> 2

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<212> DNA

<213> Unknown (Unknown)

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gggtggggga gggagtggaa tgggtgggtt cccggaagcc acgggtaacc ttctcgatgc 60

cgcagctcag gagttccacc ctacggtctg tgccccctat cctctacagc cgcttccgca 120

acagctatac tgcccccacc catatccagc catgccggtg cctccgccgc cgcaaatagc 180

catgttacag ccagtgcctc cgatggcgat ggccatggcg ccgcagccgg ggtacacctt 240

gccaacgacg acgccggtgg tcaatggccc gtcgagccgc gtcgtggtgc tgggccttgt 300

cccgccgcac gcgcaggagg ccgacgtggc gcaggcgatg gcgccattcg gcgcgatccg 360

ctcggtcgac gcgtgcgcgg tggcgtccga gggcgtggcc accgtccatt tcttcgacat 420

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cctcgggcag ctctacgcgg cggccgccgt agccccggcg tgggctcctg caccgacgcc 540

ccaggcctgg gactggcccc accccaacga cgacggccgc ggcctcgtcc tcgggcacgc 600

cgtgtgggcc cacttcgcca ccggcgccga cgacggcgac aaccgcggct ccctggtggt 660

cctgagcccc ctgcccggcg tctcggtcgc tgacctccgc caagtcttcc aggccttcgg 720

tacgcgccac cgaccgagcc gaccaaccag gcatttcgtt ttcccacgct cgtttcttgt 780

gtttcccgca gcaattgtat tggcccgtcc tcgtgttcgc aggggacttg aaggatgtga 840

gggagtcggc gcagcggccc agccacaagt tcgtggactt cttcgacacg cgcgacgccg 900

cgcgcgcgct cgccgagctc aacggccagg agcttttcgg ccgccgcctc gtcgtcgagt 960

tcacgcgccc ttccggcccc gggccccgca ggtaaacaac aacgcaattt cagtcagcta 1020

gccttcccat caccctgtgg gcggctgaat ttttgccgtg tgcctgtcgg tcctaggcgc 1080

gggtacgcac cccaccagca ccggcccacc gcgccgactc cgccgaggct tcaagcgacg 1140

tggcgaccgt cccaaccgac gtcgtctcag ccgccggcat cctcgtcgtc gtccggttcc 1200

gtaagggcga gggaaggagt ggtgcttctg aggaggagct cctgtaagtc tagcgcgggc 1260

agcgaccagt cgtccaaggg aggcaatgcc ggaacgagcc atgagcgcaa gaccaagggc 1320

ggcaagatcg tggtggcggc ggcggcggca tcctcgtcga ccccgacagc gtccgggaag 1380

caaacccaga aaggcgtcgg gagcagcggc ggcgggagct ggaaaggacg aaagagcggg 1440

tgggaggcgc gcttcctgtt caaggagccc gaggccggcg gcggcgccga cacgcaagca 1500

acgccggctt cggagatgga tacgaggacc accgtcatga tcaggaacat accgaacaag 1560

tacaggtatc cctgccaatc tccacgtttg ttgcaacaat gctatatttc atgggcgacg 1620

cacagaattg cgattctgct agctgatgcc cgaggcgttt gggtcgcagc cagaagctgc 1680

tgctcaacat gctggacaac cactgcatcc aatccaacga gtggatcgtg gcgagcggcg 1740

aggagcagcc cttctccgcc tacgatttcg tctacctccc catagatttc aagtgagttg 1800

cgcgctcggc ttccttctca cataattgca tttagctgtt cgcattgact tatattttat 1860

ggtgcccaat gcaagaacac acacaagctt cccagcctga attcggttgt tttcctggga 1920

atttgttgcc ttgaataatg gttggagtgg gaaatgtggg cgggggggtg ggaggcgagg 1980

agctggctgt ggaagcgtga gcttcaggat cgaggtatct tatctttggg tgggtagagt 2040

acagtgcagc aggcatagat gggctatggc gttactagcc caaatggaaa taaatttggc 2100

agcggcaagc aagaccatgc cagatccttc tcgttttgat gcccccccat ggattctttg 2160

gttgcatgcg cgcatctgct ggctgctgct gccgctgccg atggcgaggg agccggcctg 2220

ccttcctttc tcgcggctgc tgccgcccct gggatcgcct ccctgcctgc ctttcttgct 2280

ttgcacagcc gtgcacttcc ataacgaggc caacacatac aaaaggtgcg gcaccgatgg 2340

ggttgtagtg ttgagactag acagtagaga tagacgctta gctatggaga tggagctgtg 2400

aaaaaaaaag agttgcatga gtatgaaaag ggggaaaaag tcagcacttt cgttctaaag 2460

agaaaatttg aacttcagat caaagtagtt ttctatatgc aaacaaaatg tatgctttca 2520

agtttgctta tttaaagcac ttgcttttat atataccgcc ggcagctttt ctcttgcacg 2580

aagcaagttt gagaaaagac ctatcaatgc ccaatgacga gaggacatta gtggctgtca 2640

aaaggcatgc agcttcaaac atactcatgt gtaaagcagc tgcagcataa tgccaaagtt 2700

tttttttgat tgcaagtctt tttagttatt gttcctccat gccattacca ttctgaattt 2760

cttgcccttg catgaaatga gaatttttct tccttatgca aaaatgaaaa aaaacaaaaa 2820

aatgcagcaa caagtgtaat gtgggctacg gcttcgtcaa cctgacatcg ccggaggctg 2880

ccgtgcggct gtacaaggcg ttccacaagc agccatggga ggtgtacaac tcgcgcaaga 2940

tctgccaagt gacatacgcg cgcgtacaag tacgtactcc actcctgcag ctgcccttct 3000

cctacatcta catccgtcga agacatttta atgctacgta cgtacttatt gctcacatgt 3060

caatgttaca attgtgcggc ggcgcagggc ctggaagcgc tgaaggagca cttcaagaac 3120

tccaagttcc cgtgcgacag cgacgagtac ctgcccgtgg cgttctcgcc ggcgcgcgac 3180

ggcaaggagc ttacggatcc agtgcccatc gtgggccgct cgcccgcggc gtcgtccgcg 3240

tcgtcgcctc ccaagagccg ggcggctagc gtggaccggc ttgggcagga gctgatgccg 3300

gcgccgtcgt catccgcgga cggcgcgtcg tcgaccacta cgtccaccca cgcgccgtcc 3360

gaacacgacg aggaggagga ggagggagac atcaggctcg caggcgagct gcggcggctt 3420

ggctacgacg actagctggc tccgatccta gctgcagcta ggcgggcgac gcctagctca 3480

gcaacgatcg gcatggcgtg gcgtcgcttc gagaagcgcg gaggcagtag tgacttcggc 3540

caagcttttc acagatccgt agcgctcgct cctgcacgcg caggcgaagc aagtgaagtg 3600

gactgggact tgactttgga gaagcaaaag tgagctgatg ttcgttggtt cgtagacgct 3660

gcgcacagca ggtgtggagt cgtgttcttt tggattttgg tttgaagttt cctactactt 3720

gtttctttga aacttggagc tatagctagc ttaggcttag ctaggactgc tgggagggga 3780

aagggcatgc aggggactat caattcgtgg ttcgtcatgc gctcgttctt tacattaaaa 3840

taagattgtt attagtacag ctttacaata 3870

<210> 3

<211> 172

<212> DNA

<213> Unknown (Unknown)

<400> 3

cggtgcgatg ccccccatcg taggtgaagg tggaaattaa tgatccatct tgagaccaca 60

ggcccacaac agctaccagt ttcctcaagg gtccaccaaa aacgtaagcg cttacgtaca 120

tggtcgataa gaaaaggcaa tttgtagatg ttaacatcca acgtcgcttt ca 172

<210> 4

<211> 18041

<212> DNA

<213> Unknown (Unknown)

<400> 4

ctcgacgcat gcctacagtg cagcgtgacc cggtcgtgcc cctctctaga gataatgagc 60

attgcatgtc taagttataa aaaattacca catatttttt ttgtcacact tgtttgaagt 120

gcagtttatc tatctttata catatattta aactttactc tacgaataat ataatctata 180

gtactacaat aatatcagtg ttttagagaa tcatataaat gaacagttag acatggtcta 240

aaggacaatt gagtattttg acaacaggac tctacagttt tatcttttta gtgtgcatgt 300

gttctccttt ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta 360

catccattta gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt 420

ttattctatt ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta 480

ataatttaga tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta 540

agaaattaaa aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt 600

aaacgccgtc gacgagtcta acggacacca accagcgaac cagcagcgtc gcgtcgggcc 660

aagcgaagca gacggcacgg catctctgtc gctgcctctg gacccctctc gagagttccg 720

ctccaccgtt ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg agcggcagac 780

gtgagccggc acggcaggcg gcctcctcct cctctcacgg cacggcagct acgggggatt 840

cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct 900

ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc 960

ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc 1020

ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact 1080

tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac 1140

acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg 1200

gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg 1260

tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg 1320

tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg 1380

ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt 1440

ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga 1500

aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga 1560

tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct 1620

agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat 1680

gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga 1740

taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc 1800

tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat 1860

tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt 1920

agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc 1980

tgttgtttgg tgttacttct gcaggtcgac tctagaggat ctaccatggc caccgccgcc 2040

gccgcgtcta ccgcgctcac tggcgccact accgctgcgc ccaaggcgag gcgccgggcg 2100

cacctcctgg ccacccgccg cgccctcgcc gcgcccatca ggtgctcagc ggcgtcaccc 2160

gccatgccga tggctccccc ggccaccccg ctccggccgt ggggccccac cgatccccgc 2220

aagggatccg acgccctgcc cgccaccttc gacgtgatcg tgcatccagc tcgcgaactc 2280

cgcggcgagc ttcgcgctca gccatccaag aactacacca ctcgctacct cctcgccgct 2340

gccctcgctg agggcgagac ccgcgtggtg ggcgtggcta cctctgagga cgccgaggcc 2400

atgctccgct gcctccgcga ctggggcgct ggcgtggagc ttgtgggcga tgacgccgtg 2460

atccgcggtt tcggcgctcg cccacaggcc ggtgtgaccc tcaacccagg caacgctgcc 2520

gcggtggccc gcctcctcat gggcgtggcc gctctcacct ctggcaccac tttcgtgacc 2580

gactacccgg actccctcgg caagcgccct cagggcgacc tccttgaggc cctcgaacgc 2640

ctcggtgcct gggtgtcctc caacgacggt cgcctcccga tctccgtgtc cggcccagtg 2700

cgcggtggca ccgtggaggt gtccgccgag cgctcctccc agtacgcctc cgccctcatg 2760

ttcctcggcc ctctcctccc ggacggactc gaactccgcc tcaccggcga catcaagtcc 2820

cacgctccgc tccgccagac actcgacacc ctctctgact tcggcgtgcg cgccactgcc 2880

tccgacgacc tccgccgcat ctccatcccg ggtggccaga agtaccgccc aggccgcgtg 2940

ctcgtgccgg gcgactaccc gggctccgct gccatcctca ccgccgctgc cctcctccca 3000

ggcgaggtgc gcctctctaa cctccgcgag cacgacctcc agggcgagaa ggaggccgtg 3060

aacgtgctcc gcgagatggg cgctgacatc gtgcgcgagg gcgataccct caccgtgcgc 3120

ggtggccgcc ctctccacgc cgtgactcgc gacggcgatt ccttcaccga cgccgtgcaa 3180

gccctcaccg ccgctgctgc cttcgccgag ggcgacacca cctgggagaa cgtggccact 3240

ctccgcctca aggagtgcga ccgcatctct gacacccgcg ctgagcttga gcgcctcggc 3300

ctccgcgcac gcgagaccgc cgactctctc tccgtgactg gctctgctca cctcgctggt 3360

ggcatcaccg ccgacggcca cggcgaccac cgcatgatca tgctcctcac cctcctcggc 3420

ctccgcgcag acgctccact ccgcatcacc ggcgcacacc acatccgcaa gtcctaccct 3480

cagttcttcg ctcacctcga agccctcggc gctcgcttcg agtacgctga ggccaccgcc 3540

taataggagc tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag 3600

ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt 3660

atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa 3720

aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg tccgcaatgt 3780

gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag 3840

ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc 3900

agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc 3960

gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg 4020

agggtagcat gttgattgta acgatgacag agcgttgctg cctgtgatca ccgcggtttc 4080

aaaatcggct ccgtcgatac tatgttatac gccaactttg aaaacaactt tgaaaaagct 4140

gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt cgtcttgtta 4200

taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa taataaatgg 4260

ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag 4320

atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat 4380

atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg 4440

acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc 4500

atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt 4560

atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct 4620

ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg 4680

aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagaag 4740

acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag 4800

aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca 4860

aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg 4920

ccttctgcgt ccggtcgatc agggaggata tcggggaaga acagtatgtc gagctatttt 4980

ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg 5040

aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt tcgttccact 5100

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 5160

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 5220

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 5280

ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 5340

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 5400

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 5460

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 5520

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 5580

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 5640

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 5700

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 5760

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 5820

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 5880

gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 5940

tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 6000

agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca 6060

cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 6120

acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 6180

acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac ggcgcggctt 6240

gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg ccagcggccg 6300

cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg gtaggcgctt 6360

tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg cgggttttaa 6420

gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc tcttttatat 6480

cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca atgtacgggt 6540

tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca ggaaagagac 6600

cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc gtacattagg 6660

aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg atcgggccag 6720

cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg atcagcttgc 6780

gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg tagatcgtct 6840

tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg tagagaaaac 6900

ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg tccgggttct 6960

tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg tactccggcc 7020

gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc tcggataccg 7080

tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc gtggtgttta 7140

accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct cgccggcaga 7200

acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct cccttccctt 7260

cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc aggtcgtaat 7320

cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg tctggaagct 7380

cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa acggccacgt 7440

ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg aaaaaatctg 7500

gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc ggcggttgcc 7560

gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg cgtgcttctg 7620

cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc aggtcatcac 7680

ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgtcacgc cgattcctcg 7740

ggcttggggg ttccagtgcc attgcagggc cggcagacaa cccagccgct tacgcctggc 7800

caaccgcccg ttcctccaca catggggcat tccacggcgt cggtgcctgg ttgttcttga 7860

ttttccatgc cgcctccttt agccgctaaa attcatctac tcatttattc atttgctcat 7920

ttactctggt agctgcgcga tgtattcaga tagcagctcg gtaatggtct tgccttggcg 7980

taccgcgtac atcttcagct tggtgtgatc ctccgccggc aactgaaagt tgacccgctt 8040

catggctggc gtgtctgcca ggctggccaa cgttgcagcc ttgctgctgc gtgcgctcgg 8100

acggccggca cttagcgtgt ttgtgctttt gctcattttc tctttacctc attaactcaa 8160

atgagttttg atttaatttc agcggccagc gcctggacct cgcgggcagc gtcgccctcg 8220

ggttctgatt caagaacggt tgtgccggcg gcggcagtgc ctgggtagct cacgcgctgc 8280

gtgatacggg actcaagaat gggcagctcg tacccggcca gcgcctcggc aacctcaccg 8340

ccgatgcgcg tgcctttgat cgcccgcgac acgacaaagg ccgcttgtag ccttccatcc 8400

gtgacctcaa tgcgctgctt aaccagctcc accaggtcgg cggtggccca tatgtcgtaa 8460

gggcttggct gcaccggaat cagcacgaag tcggctgcct tgatcgcgga cacagccaag 8520

tccgccgcct ggggcgctcc gtcgatcact acgaagtcgc gccggccgat ggccttcacg 8580

tcgcggtcaa tcgtcgggcg gtcgatgccg acaacggtta gcggttgatc ttcccgcacg 8640

gccgcccaat cgcgggcact gccctgggga tcggaatcga ctaacagaac atcggccccg 8700

gcgagttgca gggcgcgggc tagatgggtt gcgatggtcg tcttgcctga cccgcctttc 8760

tggttaagta cagcgataac cttcatgcgt tccccttgcg tatttgttta tttactcatc 8820

gcatcatata cgcagcgacc gcatgacgca agctgtttta ctcaaataca catcaccttt 8880

ttagacggcg gcgctcggtt tcttcagcgg ccaagctggc cggccaggcc gccagcttgg 8940

catcagacaa accggccagg atttcatgca gccgcacggt tgagacgtgc gcgggcggct 9000

cgaacacgta cccggccgcg atcatctccg cctcgatctc ttcggtaatg aaaaacggtt 9060

cgtcctggcc gtcctggtgc ggtttcatgc ttgttcctct tggcgttcat tctcggcggc 9120

cgccagggcg tcggcctcgg tcaatgcgtc ctcacggaag gcaccgcgcc gcctggcctc 9180

ggtgggcgtc acttcctcgc tgcgctcaag tgcgcggtac agggtcgagc gatgcacgcc 9240

aagcagtgca gccgcctctt tcacggtgcg gccttcctgg tcgatcagct cgcgggcgtg 9300

cgcgatctgt gccggggtga gggtagggcg ggggccaaac ttcacgcctc gggccttggc 9360

ggcctcgcgc ccgctccggg tgcggtcgat gattagggaa cgctcgaact cggcaatgcc 9420

ggcgaacacg gtcaacacca tgcggccggc cggcgtggtg gtgtcggccc acggctctgc 9480

caggctacgc aggcccgcgc cggcctcctg gatgcgctcg gcaatgtcca gtaggtcgcg 9540

ggtgctgcgg gccaggcggt ctagcctggt cactgtcaca acgtcgccag ggcgtaggtg 9600

gtcaagcatc ctggccagct ccgggcggtc gcgcctggtg ccggtgatct tctcggaaaa 9660

cagcttggtg cagccggccg cgtgcagttc ggcccgttgg ttggtcaagt cctggtcgtc 9720

ggtgctgacg cgggcatagc ccagcaggcc agcggcggcg ctcttgttca tggcgtaatg 9780

tctccggttc tagtcgcaag tattctactt tatgcgacta aaacacgcga caagaaaacg 9840

ccaggaaaag ggcagggcgg cagcctgtcg cgtaacttag gacttgtgcg acatgtcgtt 9900

ttcagaagac ggctgcactg aacgtcagaa gccgactgca ctatagcagc ggaggggttg 9960

gatcaaagta ctttgatccc gaggggaacc ctgtggttgg catgcacata caaatggacg 10020

aacggataaa ccttttcacg cccttttaaa tatccgttat tctaataaac gctcttttct 10080

cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg aaggcgggaa 10140

acgacaatct gatccaagct caagctgctc tagcattcgc cattcaggct gcgcaactgt 10200

tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 10260

gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 10320

acggccagtg ccaagcttcc cctgaatttc agtctctcta gctagtggct aaggccatct 10380

cttatttggt tcagatgaca gtaattcatc caggtctcca agttctagga ttttcagaac 10440

tgcaacttat tttatcaagg aatctttaaa catacgaaca gatcacttaa agttcttctg 10500

aagcaactta aagttatcag gcatgcatgg atcttggagg aatcagatgt gcagtcaggg 10560

accatagcac aagacaggcg tcttctactg gtgctaccag caaatgctgg aagccgggaa 10620

cactgggtac gttggaaacc acgtgatgtg aagaagtaag ataaactgta ggagaaaagc 10680

atttcgtagt gggccatgaa gcctttcagg acatgtattg cagtatgggc cggcccatta 10740

cgcaattgga cgacaacaaa gactagtatt agtaccacct cggctatcca catagatcaa 10800

agctgattta aaagagttgt gcagatgatc cgtggcacct ccgcctcgcc cgaccctttt 10860

agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 10920

cgagtcggtg ctttttttca agcttgccta cagtgcagcg tgacccggtc gtgcccctct 10980

ctagagataa tgagcattgc atgtctaagt tataaaaaat taccacatat tttttttgtc 11040

acacttgttt gaagtgcagt ttatctatct ttatacatat atttaaactt tactctacga 11100

ataatataat ctatagtact acaataatat cagtgtttta gagaatcata taaatgaaca 11160

gttagacatg gtctaaagga caattgagta ttttgacaac aggactctac agttttatct 11220

ttttagtgtg catgtgttct cctttttttt tgcaaatagc ttcacctata taatacttca 11280

tccattttat tagtacatcc atttagggtt tagggttaat ggtttttata gactaatttt 11340

tttagtacat ctattttatt ctattttagc ctctaaatta agaaaactaa aactctattt 11400

tagttttttt atttaataat ttagatataa aatagaataa aataaagtga ctaaaaatta 11460

aacaaatacc ctttaagaaa ttaaaaaaac taaggaaaca tttttcttgt ttcgagtaga 11520

taatgccagc ctgttaaacg ccgtcgacga gtctaacgga caccaaccag cgaaccagca 11580

gcgtcgcgtc gggccaagcg aagcagacgg cacggcatct ctgtcgctgc ctctggaccc 11640

ctctcgagag ttccgctcca ccgttggact tgctccgctg tcggcatcca gaaattgcgt 11700

ggcggagcgg cagacgtgag ccggcacggc aggcggcctc ctcctcctct cacggcacgg 11760

cagctacggg ggattccttt cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat 11820

aaatagacac cccctccaca ccctctttcc ccaacctcgt gttgttcgga gcgcacacac 11880

acacaaccag atctccccca aatccacccg tcggcacctc cgcttcaagg tacgccgctc 11940

gtcctccccc cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg 12000

cccggtagtt ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc 12060

tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc 12120

cagtgtttct ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt 12180

tcatgatttt ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata 12240

tatgccgtgc acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat 12300

gatgtggtct ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg 12360

gtggatttat taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg 12420

aagatgatgg atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg 12480

atgcatatac agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg 12540

tcgttcattc gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta 12600

attttggaac tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga 12660

aatatcgatc taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat 12720

ggcatatgca gcatctattc atatgctcta accttgagta cctatctatt ataataaaca 12780

agtatgtttt ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta 12840

tatgtggatt tttttagccc tgccttcata cgctatttat ttgctatgga ctacaaggac 12900

cacgacggcg actacaagga ccacgacatc gactacaagg acgacgacga caagatggcc 12960

ccgaagaaga agaggaaggt gggcatccac ggcgtgccgg ccgccgacaa gaagtacagc 13020

atcggcctgg acatcggcac caacagcgtg ggctgggccg tgatcaccga cgagtacaag 13080

gtgccgagca agaagttcaa ggtgctgggc aacaccgaca ggcacagcat caagaagaac 13140

ctgatcggcg ccctgctgtt cgacagcggc gagaccgccg aggccaccag gctgaagagg 13200

accgccagga ggaggtacac caggaggaag aacaggatct gctacctgca ggagatcttc 13260

agcaacgaga tggccaaggt ggacgacagc ttcttccaca ggctggagga gagcttcctg 13320

gtggaggagg acaagaagca cgagaggcac ccgatcttcg gcaacatcgt ggacgaggtg 13380

gcctaccacg agaagtaccc gaccatctac cacctgagga agaagctggt ggacagcacc 13440

gacaaggccg acctgaggct gatctacctg gccctggccc acatgatcaa gttcaggggc 13500

cacttcctga tcgagggcga cctgaacccg gacaacagcg acgtggacaa gctgttcatc 13560

cagctggtgc agacctacaa ccagctgttc gaggagaacc cgatcaacgc cagcggcgtg 13620

gacgccaagg ccatcctgag cgccaggctg agcaagagca ggaggctgga gaacctgatc 13680

gcccagctgc cgggcgagaa gaagaacggc ctgttcggca acctgatcgc cctgagcctg 13740

ggcctgaccc cgaacttcaa gagcaacttc gacctggccg aggacgccaa gctgcagctg 13800

agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 13860

gccgacctgt tcctggccgc caagaacctg agcgacgcca tcctgctgag cgacatcctg 13920

agggtgaaca ccgagatcac caaggccccg ctgagcgcca gcatgatcaa gaggtacgac 13980

gagcaccacc aggacctgac cctgctgaag gccctggtga ggcagcagct gccggagaag 14040

tacaaggaga tcttcttcga ccagagcaag aacggctacg ccggctacat cgacggcggc 14100

gccagccagg aggagttcta caagttcatc aagccgatcc tggagaagat ggacggcacc 14160

gaggagctgc tggtgaagct gaacagggag gacctgctga ggaagcagag gaccttcgac 14220

aacggcagca tcccgcacca gatccacctg ggcgagctgc acgccatcct gaggaggcag 14280

gaggacttct acccgttcct gaaggacaac agggagaaga tcgagaagat cctgaccttc 14340

aggatcccgt actacgtggg cccgctggcc aggggcaaca gcaggttcgc ctggatgacc 14400

aggaagagcg aggagaccat caccccgtgg aacttcgagg aggtggtgga caagggcgcc 14460

agcgcccaga gcttcatcga gaggatgacc aacttcgaca agaacctgcc gaacgagaag 14520

gtgctgccga agcacagcct gctgtacgag tacttcaccg tgtacaacga gctgaccaag 14580

gtgaagtacg tgaccgaggg catgaggaag ccggccttcc tgagcggcga gcagaagaag 14640

gccatcgtgg acctgctgtt caagaccaac aggaaggtga ccgtgaagca gctgaaggag 14700

gactacttca agaagatcga gtgcttcgac agcgtggaga tcagcggcgt ggaggacagg 14760

ttcaacgcca gcctgggcac ctaccacgac ctgctgaaga tcatcaagga caaggacttc 14820

ctggacaacg aggagaacga ggacatcctg gaggacatcg tgctgaccct gaccctgttc 14880

gaggacaggg agatgatcga ggagaggctg aagacctacg cccacctgtt cgacgacaag 14940

gtgatgaagc agctgaagag gaggaggtac accggctggg gcaggctgag caggaagctg 15000

atcaacggca tcagggacaa gcagagcggc aagaccatcc tggacttcct gaagagcgac 15060

ggcttcgcca acaggaactt catgcagctg atccacgacg acagcctgac cttcaaggag 15120

gacatccaga aggcccaggt gagcggccag ggcgacagcc tgcacgagca catcgccaac 15180

ctggccggca gcccggccat caagaagggc atcctgcaga ccgtgaaggt ggtggacgag 15240

ctggtgaagg tgatgggcag gcacaagccg gagaacatcg tgatcgagat ggccagggag 15300

aaccagacca cccagaaggg ccagaagaac agcagggaga ggatgaagag gatcgaggag 15360

ggcatcaagg agctgggcag ccagatcctg aaggagcacc cggtggagaa cacccagctg 15420

cagaacgaga agctgtacct gtactacctg cagaacggca gggacatgta cgtggaccag 15480

gagctggaca tcaacaggct gagcgactac gacgtggacc acatcgtgcc gcagagcttc 15540

ctgaaggacg acagcatcga caacaaggtg ctgaccagga gcgacaagaa caggggcaag 15600

agcgacaacg tgccgagcga ggaggtggtg aagaagatga agaactactg gaggcagctg 15660

ctgaacgcca agctgatcac ccagaggaag ttcgacaacc tgaccaaggc cgagaggggc 15720

ggcctgagcg agctggacaa ggccggcttc atcaagaggc agctggtgga gaccaggcag 15780

atcaccaagc acgtggccca gatcctggac agcaggatga acaccaagta cgacgagaac 15840

gacaagctga tcagggaggt gaaggtgatc accctgaaga gcaagctggt gagcgacttc 15900

aggaaggact tccagttcta caaggtgagg gagatcaaca actaccacca cgcccacgac 15960

gcctacctga acgccgtggt gggcaccgcc ctgatcaaga agtacccgaa gctggagagc 16020

gagttcgtgt acggcgacta caaggtgtac gacgtgagga agatgatcgc caagagcgag 16080

caggagatcg gcaaggccac cgccaagtac ttcttctaca gcaacatcat gaacttcttc 16140

aagaccgaga tcaccctggc caacggcgag atcaggaaga ggccgctgat cgagaccaac 16200

ggcgagaccg gcgagatcgt gtgggacaag ggcagggact tcgccaccgt gaggaaggtg 16260

ctgagcatgc cgcaggtgaa catcgtgaag aagaccgagg tgcagaccgg cggcttcagc 16320

aaggagagca tcctgccgaa gaggaacagc gacaagctga tcgccaggaa gaaggactgg 16380

gacccgaaga agtacggcgg cttcgacagc ccgaccgtgg cctacagcgt gctggtggtg 16440

gccaaggtgg agaagggcaa gagcaagaag ctgaagagcg tgaaggagct gctgggcatc 16500

accatcatgg agaggagcag cttcgagaag aacccgatcg acttcctgga ggccaagggc 16560

tacaaggagg tgaagaagga cctgatcatc aagctgccga agtacagcct gttcgagctg 16620

gagaacggca ggaagaggat gctggccagc gccggcgagc tgcagaaggg caacgagctg 16680

gccctgccga gcaagtacgt gaacttcctg tacctggcca gccactacga gaagctgaag 16740

ggcagcccgg aggacaacga gcagaagcag ctgttcgtgg agcagcacaa gcactacctg 16800

gacgagatca tcgagcagat cagcgagttc agcaagaggg tgatcctggc cgacgccaac 16860

ctggacaagg tgctgagcgc ctacaacaag cacagggaca agccgatcag ggagcaggcc 16920

gagaacatca tccacctgtt caccctgacc aacctgggcg ccccggccgc cttcaagtac 16980

ttcgacacca ccatcgacag gaagaggtac accagcacca aggaggtgct ggacgccacc 17040

ctgatccacc agagcatcac cggcctgtac gagaccagga tcgacctgag ccagctgggc 17100

ggcgacaggc cgaagaagaa gaggaaggtg ggcggctaac cgatcgttca aacatttggc 17160

aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc 17220

tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat 17280

gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat 17340

agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat 17400

tgatcccccc tcgacagggt acctgcccct gaatttcagt ctctctagct agtggctaag 17460

gccatctctt atttggttca gatgacagta attcatccag gtctccaagt tctaggattt 17520

tcagaactgc aacttatttt atcaaggaat ctttaaacat acgaacagat cacttaaagt 17580

tcttctgaag caacttaaag ttatcaggca tgcatggatc ttggaggaat cagatgtgca 17640

gtcagggacc atagcacaag acaggcgtct tctactggtg ctaccagcaa atgctggaag 17700

ccgggaacac tgggtacgtt ggaaaccacg tgatgtgaag aagtaagata aactgtagga 17760

gaaaagcatt tcgtagtggg ccatgaagcc tttcaggaca tgtattgcag tatgggccgg 17820

cccattacgc aattggacga caacaaagac tagtattagt accacctcgg ctatccacat 17880

agatcaaagc tgatttaaaa gagttgtgca gatgatccgt ggccatcgcc caacctaggg 17940

cgcttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 18000

gtggcaccga gtcggtgctt ttttggtacc gagctcgaat t 18041

<210> 5

<211> 85

<212> DNA

<213> Unknown (Unknown)

<400> 5

tcggactcgg gctaagtcct agaagacgac gaactccgct tcgcccgacc ccagggctcg 60

gactccgccc tggcctcagc cgacg 85

<210> 6

<211> 60

<212> DNA

<213> Unknown (Unknown)

<400> 6

ccagccacgt caacaggagg cgccatcatc accctacccc gagctgactc gggccgcagg 60

<210> 7

<211> 70

<212> DNA

<213> Unknown (Unknown)

<400> 7

caagtcggtc gtcttaagaa atcaacgcac ttcaacagta ttgagccgtc tggtgtcaaa 60

tgagacttgc 70

<210> 8

<211> 80

<212> DNA

<213> Unknown (Unknown)

<400> 8

ttctttttca gtgcaacaag ggatgtagag gtgccatggt ttgggtaagc actcatttct 60

gtggtttata gttcgtgagt 80

<210> 9

<211> 80

<212> DNA

<213> Unknown (Unknown)

<400> 9

ctgcacaggc gcggacggtc cgcggccagg ggccggacgg tctgcgacct ggcgcagggc 60

ttagggtttc ctgcttgacg 80

<210> 10

<211> 80

<212> DNA

<213> Unknown (Unknown)

<400> 10

ccgacctaga ctcctatcga tatagagtcg aagagaagcg gagaatttgg ggattggaag 60

gctaaactag aactactcct 80

<210> 11

<211> 80

<212> DNA

<213> Unknown (Unknown)

<400> 11

gaatctattt tctctctcct caagttaagt tgagactcct gagtttattg tatctttcat 60

taactctagt tttttgtatt 80

<210> 12

<211> 80

<212> DNA

<213> Unknown (Unknown)

<400> 12

ggggtaattc aaacctcacg acaattttaa tatttgaagc ttataatgat caatttttta 60

ctctaaagta ttcttaaata 80

Claims (10)

1. A method for increasing corn biomass, characterized in that the method comprises: inserting a regulatory sequence into the upstream or downstream of a target gene of a corn genome at a fixed point to obtain corn with improved biological quantity; the target gene is PLA1 gene or TE1 gene, and the regulatory sequence is shown in SEQ ID NO. 3.

2. The method for improving the biomass of the corn as claimed in claim 1, characterized in that the nucleotide sequence of the PLA1 gene is shown as SEQ ID NO. 1; the upstream insertion site of the PLA1 gene is between 247306010 and 247312010 bases of the maize No.1 chromosome.

3. The method for increasing the biomass of maize as claimed in claim 1, wherein the downstream insertion site of PLA1 gene is between the bases 247314293 and 247317293 of chromosome 1 of maize.

4. The method for improving the biomass of the corn as claimed in claim 1, characterized in that the nucleotide sequence of the TE1 gene is shown as SEQ ID NO. 2; the upstream insertion site of the TE1 gene is between 167811126 and 167821126 bases of the maize chromosome 3.

5. The method of claim 1 wherein the downstream insertion site of the TE1 gene is between 167824995 and 167834995 bases of chromosome 3 in maize.

6. The method for increasing corn biomass according to claim 1, wherein the regulatory sequence is inserted by: introducing a recombinant donor sequence containing a regulatory sequence and a gene editing vector containing a marker gene into a maize immature embryo by adopting a gene gun method, and screening transgenic maize without the marker gene to obtain maize with improved biological quantity; the recombination donor sequence is formed by sequentially connecting an upstream homology arm of a target gene to-be-inserted site, a regulatory sequence and a downstream homology arm of the target gene to-be-inserted site; the gene editing vector containing the marker gene comprises a Cas9 gene expression cassette, an upstream sgRNA coding sequence and a downstream sgRNA coding sequence.

7. The method for increasing the biomass of the corn as claimed in claim 1, wherein when the insertion site of the regulatory sequence is upstream of the expression frame of the target gene PLA1, an upstream homology arm shown in SEQ ID No.5 and a downstream homology arm shown in SEQ ID No.6 are designed according to the insertion site; sequentially connecting an upstream homologous arm shown by SEQ ID NO.5, a regulatory sequence shown by SEQ ID NO.3 and a downstream homologous arm shown by SEQ ID NO.6 to form a recombinant donor sequence; a sgRNA target is designed at 247310934 bases of a No.1 chromosome of corn, and is named as sgRNA1, and the sequence is as follows: acctccgcctcgcccgaccc, designing another target at 247310995bp, named sgRNA2 with sequence catcgcccaacctagggcgc; the sgRNA sequence in the gene editing vector P-G10-sgRNA-cas 9-sgRNA shown in SEQ ID NO.4 is replaced by sgRNA1, and the sgRNA sequence is replaced by sgRNA2, so that the PLA1 upstream gene editing vector P-G10-sgRNA1-cas9-sgRNA2 is obtained.

8. The method for increasing corn biomass according to claim 1, characterized in that when the regulatory sequence insertion site is downstream of the PLA1 gene expression cassette, a sgRNA target is designed at a 247314496bp site of chromosome 1 of corn, and is named sgRNA3, gttcactaaagaacttaatc; 247315566bp sites are used for designing a target, namely sgRNA4, atcaattcaagcacttaaac; designing an upstream homology arm according to the insertion site, wherein the nucleotide sequence is SEQ ID NO.7, the downstream homology arm is SEQ ID NO. 8; and sequentially connecting an upstream homologous arm shown by SEQ ID NO.7, a regulatory sequence shown by SEQ ID NO.3 and a downstream homologous arm shown by SEQ ID NO.8 to form a recombinant donor sequence.

9. The method for increasing the biomass of the corn as claimed in claim 1, wherein when the insertion site is upstream of an expression cassette of a TE1 gene, an sgRNA target is designed at 167822232bp of a No.3 chromosome of the corn, and is named as sgRNA5, and the sequence is as follows: aggtgttgtctaggcttggc, designing another target at 167812349bp, named sgRNA6 with sequence gtcggacgatccgcgcctat; designing an upstream homology arm according to the insertion site, wherein the nucleotide sequence is SEQ ID NO.9, and the nucleotide sequence of a downstream homology arm is SEQ ID NO. 10; and sequentially connecting an upstream homologous arm shown by SEQ ID NO.9, a regulatory sequence shown by SEQ ID NO.3 and a downstream homologous arm shown by SEQ ID NO.10 to form a recombinant donor sequence.

10. The method for increasing the biomass of the corn as claimed in claim 1, wherein when the insertion site is downstream of an expression cassette of a TE1 gene, an sgRNA target is designed at 167825705bp of a No.3 chromosome of the corn, and is named as sgRNA7, and the sequence is as follows: ttcttataaataaatatttt, designing another target at 167825814bp, named sgRNA8 with sequence cttgaagtgagatcttaaat; designing an upstream homology arm according to the insertion site, wherein the nucleotide sequence is SEQ ID NO.11, the downstream homology arm is SEQ ID NO. 12; and sequentially connecting an upstream homologous arm shown by SEQ ID NO.11, a regulatory sequence shown by SEQ ID NO.3 and a downstream homologous arm shown by SEQ ID NO.12 to form a recombinant donor sequence.

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