CN107353332A - A kind of rice chloroplast developmental regulation Gene A HS1 and its coding protein and application - Google Patents

Abstract

The invention discloses a kind of protein of rice chloroplast developmental regulation Gene A HS1 codings, there is Seq ID No：Amino acid sequence shown in 2；The invention also discloses a kind of gene for encoding above-mentioned protein, there is Seq ID No：Genome nucleotide sequence shown in 1；The invention also discloses the plant expression vector containing said gene, host cell, and above-mentioned protein, gene, plant expression vector, host cell are in the developmental application of regulation and control plant chloroplast and the method for improvement rice leaf color.The present invention is cloned into AHS1 genes in rice first using map-based cloning, pass through the functional analysis to AHS1 genes, it further specify that the genetic mechanism that plant particularly grass Development of Chloroplasts and leaf color are formed, to improve the photosynthetic efficiency of crops, improve increasing production of rice potentiality and lay a good foundation.

Description

A kind of rice chloroplast developmental regulation Gene A HS1 and its coding protein and application

Technical field

The invention belongs to plant genetic engineering field.Specifically, the present invention relates to one kind to utilize map-based cloning gram Precititation rice AHS1 (Albino in Heat Stress1) gene, and identify using transgene complementation test the work(of the gene Energy；Also relate to be regulated and controled using the gene pairs rice chloroplast growth course and leaf color, to improve crops Photosynthetic efficiency, improve increasing production of rice potentiality.

Background technology

Chloroplaset be it is distinctive in green plants mesophyll cell can carry out photosynthetic organelle, it is distributed mainly on In the cortical cell of mesophyll cell and young stem.In ontogeny, chloroplaset comes from proplastid, is to develop into leaf by proplastid Green body.In the growth course of chloroplaset, the formation of the thylakoid membrane in it and the accumulation of chlorophyll are two complementary Process.When the gene related to Development of Chloroplasts is undergone mutation, it will the normal development of chloroplaset is had influence on, so as to influence leaf Green plain biosynthesis, the pigment ratio in chlorophyll is caused to change and produce leaf color mutant.And leaf color is to influence rice light Cooperation is used and the important character of yield, is occupied an important position in terms of rice yield and breed improvement is improved.

Rice chloroplast is the important place of synthesis of organic substance matter, by delaying the aging of blade and extending photosynthetic organs Function, dry-matter accumulation can be increased；Especially in the late growth stage of rice, by extending the photosynthesis time of plant leaf, The potential of increasing production of rice can be improved by accumulating more organic substances.There is a kind of important leaf color mutant in rice -- often Greenery, it is a kind of evergreen mutant of functional form, and its main phenotypic characteristic contains for growth anaphase blade Senescence, chlorophyll Amount and photosynthetic capacity keep constant.Therefore, effective utilization of leaf color gene, can not only improve the production capacity of rice, also for Super rice breeding from now on provides new approaches and methods.

The content of the invention

It is an object of the invention to provide it is a kind of can influence rice chloroplast development and the protein of leaf color, its gene and Using.

To achieve the above object, the invention provides a kind of albumen of rice chloroplast developmental regulation Gene A HS1 codings Matter, the protein have the sequence shown in (A) or (B)：(A)Seq ID No：Amino acid sequence shown in 2 or Fig. 8；(B) exist (A) added in the amino acid sequence limited and/or substitute and/or lack one or more amino acid and there is identical function The protein as derived from (A).

Present invention also offers a kind of gene for encoding above-mentioned protein, the gene has the sequence shown in (a) or (b)： (a)Seq ID No：Genome nucleotide sequence shown in 1 or Fig. 7；(b) in the nucleotide sequence shown in (a) addition and/or Substitute and/or the codified that lacks one or more nucleotides and generate has the protein of rice chloroplast developmental regulation function Mutator, allele or derivative.

Present invention also offers a kind of plant expression vector for including said gene.The plant expression vector is preferred PCambia1300 expression vectors.

Present invention also offers a kind of host cell, the host cell contains said gene sequence.The host cell is big Coli cell, agrobatcerium cell or plant cell.

Present invention also offers a kind of method for improveing rice leaf color, by said gene rice transformation cell, then will convert Rice cell afterwards is cultivated into plant.

Present invention also offers above-mentioned protein, gene, plant expression vector, host cell in regulation and control plant chloroplast hair Application in educating.The plant is preferably grass, in particular rice.

It is further elaborated with：It is an object of the invention to provide one kind from rice mutant albino in heat The new gene AHS1 cloned in stress1, such as Fig. 7 and Seq ID No：DNA sequence dna shown in 1, also include and Seq ID No：1 The gene order of shown DNA sequence dna at least 70% homology；It is additionally included in Seq ID No：In 1 add and/or substitute and/ Or mutator, allele or the derivative for lacking one or more nucleotides and generating, the sequence with identical function The purpose of the present invention can be reached.Seq ID No in the present invention：Protein shown in 2 and Fig. 8 belongs to triangular shape pentapeptide and includes egg In vain, wherein carrying out one or more replacements, the purpose of the present invention can also be reached by inserting or lack obtained functional analogue.

It is a further object to provide a kind of method that efficient Plant Transformation is carried out with AHS1 genes, specifically Say, the invention provides with Seq ID No：The gene of sequence shown in 1 and Fig. 7 or the carrier of Gene Partial fragment, wherein, such as PCambia1300-AHS1 shown in Fig. 4, the carrier can express above-mentioned nucleotide sequence coded polypeptide or its homologous class Like thing.

Present invention also offers a kind of method that rice leaf color is influenceed using plant expression vector conversion plant cell. Specifically plant cell is converted using plant expression vector to influence the method for rice chloroplast development.

Realize that the particular technique step of the present invention is as follows：

(1) mutant ahs1 separation and genetic analysis：

The rice seedling albino mutant ahs1 of the present invention comes from japonica rice variety Nipponbare (Oryza sativa L.cv Nipponbare) it is mutated caused by EMS (Ethyl Methyl Sulfonate) mutagenesis.It is real by the reciprocal cross with wild type Test, it was demonstrated that the mutant is controlled by Recessive genes, as shown in Figure 1.

(2) mutant ahs1 is compared with wild type mesophyll cell chloroplaset and glume parenchyma cell chloroplast structure：

Compared with wild type, thylakoid structure unobvious or number are decreased obviously in mutant ahs1 mesophyll cell chloroplasets (shown in such as Figure 1B, 1C)；Compared with wild type, without ripe or complete chloroplast structure in mutant ahs1 glume parenchyma cells (such as Fig. 2 B, shown in 2C).

(3) map based cloning AHS1 genes：

1) AHS1 first positioning：

In order to separate AHS1 genes, the present invention constructs a target group first, by mutant ahs1 and rice variety Local No. 1 hybridization combines F in platform₂Target group.Again by the method for map based cloning, being marked using STS, SSR equimolecular will AHS1 sites Primary Location is in the region between the 5th chromosome marks between 5-11 and A5-9 (Fig. 3).

2) AHS1 finely positioning：

Develop new STS marks AHS1 to be accurately located on BAC OJ1268_B08, between K5-13 and K5-27 mark In the range of 22kb (Fig. 3), candidate gene is speculated by analyzing this section ORFs (ORF).

3) identification and functional analysis of AHS1 genes：

Pass through transgenic technology, the results showed that present invention obtains the transgenic paddy rice for making mutant recover normal phenotype (Fig. 5, Fig. 6), it was demonstrated that of the invention correctly to have cloned AHS1 genes.

In summary, the present invention utilizes rice seedling albefaction ahs1 mutant, by map-based cloning first in rice In be cloned into AHS1 genes, one triangular shape pentapeptide of the gene code includes albumen, and adjusting and controlling rice blade and glume middle period are green Body early development, so as to influence leaf color and glume color.By the functional analysis to AHS1 genes, plant further specify that The genetic mechanism that thing particularly grass Development of Chloroplasts and leaf color are formed, to improve the photosynthesis of crops effect Rate, improve increasing production of rice potentiality and lay a good foundation.

Brief description of the drawings

The seedling stage phenotypic map (A) and blade and blade that Fig. 1 is rice chloroplast developmental defect material ahs1 and wild type material are green Body micro-structure diagram (B and C).

Fig. 2 is rice chloroplast developmental defect material ahs1 and wild type material fringe portion phenotypic map (A) and glume chloroplaset Micro-structure diagram (B and C).

Fig. 3 is that the finely positioning of AHS1 genes and clone scheme.

Fig. 4 is pCAMBIA1302-AHS1 Vector maps.

Fig. 5 is transgenosis complementation T₁For rice plant and ahs1 mutant plants phenotypic maps.A left side, ahs1 mutant seedlings； The right side, transgenosis complementation T₁For seedling.

Fig. 6 is transgenosis complementation T₁For rice plant and ahs1 mutant fringe portion phenotypic maps.A left side, ahs1 mutant fringe portions； The right side, transgenosis complementation T₁For fringe portion.

Fig. 7 is the DNA nucleotide sequence figure of AHS1 genes.

Fig. 8 is the amino acid sequence figure of AHS1 gene codes.

Embodiment

Following examples further illustrate present disclosure, but should not be construed limitation of the present invention.Do not carrying on the back In the case of from spirit of the invention and essence, the modifications or substitutions made to the inventive method, step or condition belong to this hair Bright scope.Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art. Unless otherwise specified, biochemical reagents used in embodiment, carrier, consumptive material etc. are commercially available products.

The rice chloroplast developmental regulation Gene A HS1 of embodiment 1. clone

(1) rice material：

Rice mutant ahs1 (albino in heat stress1), its original wild material are japonica rice variety Nipponbare (Oryza sativa L.cv Nipponbare).As shown in figure 1, mutant ahs1 wheat seedings albefaction (Figure 1A)；With it is wild Type is compared, and ahs1 Development of Chloroplasts is abnormal (Figure 1B, 1C).At heading stage, mutant ahs1 glumes show albefaction phenotype, mutation It is visible (Fig. 2 B, 2C) without complete chloroplaset in body ahs1 glumes.

(2) analysis and target group：

Local No. 1 is hybridized in homozygous ahs1 mutant and wild type rice variety platform, F₁Generation selfing, obtains F₂Group Body, and 2104 plants of ahs1 phenotype individuals are therefrom selected as target group.In every plant of tender leaf for taking 1 gram or so of seedling stage, for carrying Take plant genomic DNA.

(3) positioning of AHS1 genes：

Using the ahs1 phenotype individuals chosen from target group, the microcommunity of composition carries out ssr analysis, according to announcement The molecular genetic linkage map that japonica rice and long-grained nonglutinous rice create, choose the SSR primers that approaches uniformity is distributed on each bar chromosome and enter performing PCR expansion Increase, using following amplification program：94 DEG C of pre-degenerations 4 minutes；94 DEG C be denatured 30 seconds, 55 DEG C anneal 30 seconds, 72 DEG C extend 30 seconds, 35 Individual circulation；Last 72 DEG C re-extend 10 minutes.PCR primer dyes through the separation of 5% agarose gel electrophoresis and Ethidum Eremide, detection The polymorphism of PCR primer, by AHS1 Primary Locations between No. 5 the short arm of a chromosome 5-11 and A5-9 are marked.What is just positioned On the basis of continue to design SSR and STS marks, most at last AHS1 is accurately located at No. BAC as common 22kb on OJ1268_B08 sections Within the scope of, the molecular labeling on both sides is K5-13 and K5-27 (Fig. 3), and is isolated with K5-29.Primer sequence is as follows：

5-11 F：GCTCTCCTGTGGGTTTTCAG R：CATGGTGCTCCTACTGGTTG

A5-9 F：ACTTACATCTGAGGTGCATA R：GCATTGCAGATTACAGATAC

K5-13 F：TCGTCGCACGCGAGATTTT R：ACACCGAACTGGGGCTGT

K5-27 F：GCGTCTCACCTAGCACTAT R：CGTCGCTCTATTTATCACAG

K5-29 F：CGATTTTGATGAACCAGAT R：TGCTCTCTCAGACTAAATG

(4) predictive genes and sequencing analysis：

According to the result of finely positioning, according to Rice Automated Annotation System in the range of 22kb (http://RiceGAAS.dna.affrc.go.jp) prediction, find to share 4 candidate genes in this section, according to two Mark remaining restructuring number of individuals and to isolate mark, we devise the sequencing primer of each gene, using PCR method respectively from Each candidate gene progress sequencing analysis are amplified in ahs1 and wild-type variety Nipponbare genome.It was found that wherein 1 candidate In 1 DNA fragmentation of gene, product and the wild-type variety Nipponbare of mutant ahs1 amplifications relatively have 1 base to replace.Will Above-mentioned sequencing procedure repeated authentication twice, draws identical result.Therefore, the candidate gene is named as AHS1.According to BAC The gene annotation information of OJ1268_B08 sequences is cloned, predicts that the candidate gene encodes a triangular shape pentapeptide and includes albumen.

Embodiment 2.pCAMBIA1300-AHS1 plant expression vector constructions

According to rice varieties Nipponbare (the Oryza sativa L cv.Nipponbare) full-length genome provided in NCBI Sequence, the specificity amplification primer of design amplification AHS1 candidate gene full length sequences, and expressed according to the pCambia1300 of selection The feature of carrier and AHS1 candidate gene sequences, in specific primer both ends addition specific cleavage site (Fig. 4).Specific design Primer be：The end addition EcoRI restriction enzyme sites of forward primer (P1F) 5 ' (GAATTC), the addition of the end of reverse primer (P1R) 5 ' Sse83871 restriction enzyme sites (CCTGCAGG), primer sequence is as follows：

P1F forward primers：5’-CGGAATTCACTCCGATTTCCGTCTCTT-3’

P1R reverse primers：5’-TGCCTGCAGGGCAGAAACGATAAGCACATA-3’

Then rice varieties Nipponbare genomic DNA is extracted, and using Nipponbare genomic DNA as template, using setting above Primer (P1F and P1R) the amplification AHS1 candidate gene full length sequences of meter amount to 4934bp：Including AHS1 genomic DNAs total length and Downstream sequence thereon.Using following amplification program：94 DEG C of pre-degenerations 4 minutes；98 DEG C are denatured 30 seconds, and 55 DEG C are annealed 30 seconds, 68 DEG C Extension 5 minutes, 35 circulations；Last 72 DEG C re-extend 10 minutes.The purpose fragment of PCR amplifications is reclaimed, connects ZERO BLUNT TOPO carriers convert escherichia coli DH5a competent cell, then identify and sieve by bacterium colony PCR to (Invitrogen) Positive colony is selected, and send Invitrogen companies to be sequenced positive colony.Positive colony Jing Guo sequencing identification is subjected to plasmid Extraction, the plasmid EcoRI and Sse83871 double digestions of extraction, and reclaim AHS1 complementary fragments.Simultaneously using EcoRI and Sse83871 carries out double digestion linearisation to pCambia1300, and reclaims pCambia1300 skeletons, by what is reclaimed after digestion AHS1 complementary fragments are attached with the pCambia1300 skeleton T4 ligases (being purchased from NEB companies) reclaimed after digestion, are obtained AHS1 complementation expression vector pCAMBIA1300-AHS1 (Fig. 4), pCAMBIA1300-AHS1 is expressed using electroporated method and carried Body is transferred in agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105.

Embodiment 3. is by pCAMBIA1300-AHS1 plant expression vector rice transformations

Method for transformation is contaminated using agriculture bacillus mediated Mature Embryos of Rice callus, by recombinant expression carrier pCAMBIA1300- AHS1 is transferred in Mature Embryos of Rice, and method for transformation is as follows：(1) induction of Mature Embryos of Rice callus：By the Nipponbare of maturation Seed shells, and then with 75% alcohol surface sterilization 2min, then soaks 30min with 30%NaClO solution, and is repeated once, Then cleaned 4-5 times with aqua sterilisa.Then seed is placed on inducing culture and cultivated, 26 degree of lucifuge culture evoked callus For converting.(2) co-cultivation of Rice Callus and Agrobacterium：Identification in embodiment 2 is contained into pCAMBIA1300-AHS1 The EHA105 bacterial strains of expression vector are activated, are enriched with, are resuspended, and adjust OD600=0.4-0.6.Callus is collected in In 50ml sterile centrifugation tubes, the agrobacterium suspension being resuspended is poured into, contaminates callus.After soaking 15-30min, suspension is outwelled, The callus contaminated is placed on aseptic filter paper and blots unnecessary Agrobacterium bacterium solution.Then callus is placed in and is covered with aseptic filter paper In culture dish, 26 degree of lucifuge cultures 2-3 days.(3) screening of kanamycin-resistant callus tissue：After the completion of co-cultivation, by callus be transferred to containing In the screening and culturing medium of 50mg/ml hygromycin, resistance screening under the conditions of 26-28 degree.(4) differentiation of kanamycin-resistant callus tissue：Will screening The good callus of growth conditions is placed in differential medium in culture medium, is placed in 16 hours illumination/8 hour dark, environment temperatures Under conditions of between 26-28 degree, differentiation culture, until differentiation grows seedling.(5) taking root for seedling is broken up：Seedling to be broken up During or so moon 2cm, seedling is transferred in root media, carries out culture of rootage.Grow the small transplantation of seedlings of root system in greenhouse or Transgenosis is grown in garden.Identification is carried out to plant and continuously it has been observed that compared with mutant of the same period, transgenosis Complementary T₁Normal condition (Fig. 5), transgenosis complementation T are returned to for plant seedling stage leaf color₁Also returned to for plant fringe portion glume Green (Fig. 6).

Sequence table

<110>China Paddy Rice Inst

<120>A kind of rice chloroplast developmental regulation Gene A HS1 and its coding protein and application

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gcagggttgg agttgtttct caccatgctg gagaatgagg tacaaccgaa tctcatgaca 900

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actaaagatg ccatgtcatg gactgcaatg atatcggggt atgagaaaaa tggtttccca 1140

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ggagatattg ctttatcact ctttaaccaa atggtagaaa tgggagagca tccggatgaa 1800

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gagctttttc acatgatgac tgagaaattt tcaatagttc caaatctcaa gcactatgca 1920

tgtatggtag atctattgag tcgtgttggg aaattaacag aagcttacaa cctcataaat 1980

cgaatgccta tcaaacctga tgctgcagtg tggggagcct tgttgaatgg atgccggatc 2040

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gatgttgcat atcatgttct tctgtgtgat ttatatactg atgctggcaa atgggctcaa 2160

gtggctagag tgagaaaaac catgcgagag aagggattgg agcaagataa tggatgtagc 2220

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Arg Phe Gly Phe Gly Asp Glu Val Asp Val Leu Asn Ala Leu Val Thr

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His Gly Phe Ala Val Lys Arg Gly Phe Ala Ile Asp Val Ala Phe Cys

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Ser Glu Lys Asp Val Val Ser Trp Asn Ile Met Leu Ser Gly Phe Val

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Ala His Gly Leu Gly Asp Ile Ala Leu Ser Leu Phe Asn Gln Met Val

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Glu Met Gly Glu His Pro Asp Glu Val Thr Phe Val Ala Leu Leu Cys

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Ala Cys Ser Arg Ala Gly Met Val Ile Gln Gly Trp Glu Leu Phe His

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Met Met Thr Glu Lys Phe Ser Ile Val Pro Asn Leu Lys His Tyr Ala

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Cys Met Val Asp Leu Leu Ser Arg Val Gly Lys Leu Thr Glu Ala Tyr

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Asn Leu Ile Asn Arg Met Pro Ile Lys Pro Asp Ala Ala Val Trp Gly

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Ala Leu Leu Asn Gly Cys Arg Ile His Arg His Val Glu Leu Gly Glu

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Leu Ala Ala Lys Val Ile Leu Glu Leu Glu Pro Asn Asp Val Ala Tyr

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His Val Leu Leu Cys Asp Leu Tyr Thr Asp Ala Gly Lys Trp Ala Gln

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Val Ala Arg Val Arg Lys Thr Met Arg Glu Lys Gly Leu Glu Gln Asp

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Asn Gly Cys Ser Trp Val Glu Val Lys Gly Val Thr His Ala Phe Leu

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Thr Asp Asp Glu Ser His Pro Gln Ile Lys Glu Ile Asn Val Val Leu

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His Gly Ile Tyr Glu Arg Met Lys Ala Cys Gly Phe Ala Pro Val Glu

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Arg Asp Thr Lys Gln Leu His Cys Phe Lys Asp Gly Asp Cys Ser Cys

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Gly Asp Ile Gly Tyr Gly

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<211> 19

<212> DNA

<213>Artificial sequence (K5-29 F)

<400> 11

cgattttgat gaaccagat 19

<210> 12

<211> 19

<212> DNA

<213>Artificial sequence (K5-29 R)

<400> 12

tgctctctca gactaaatg 19

<210> 13

<211> 27

<212> DNA

<213>Artificial sequence (P1F)

<400> 13

cggaattcac tccgatttcc gtctctt 27

<210> 14

<211> 30

<212> DNA

<213>Artificial sequence (P1R)

<400> 14

tgcctgcagg gcagaaacga taagcacata 30

Claims (10)

1. A kind of 1. protein of rice chloroplast developmental regulation Gene A HS1 codings, it is characterised in that：The protein has (A) Or the sequence shown in (B)：

   (A)Seq ID No：Amino acid sequence shown in 2；

   (B) add in the amino acid sequence that (A) is limited and/or substitute and/or lack one or more amino acid and have The protein as derived from (A) of identical function.
2. A kind of 2. gene for encoding protein described in claim 1, it is characterised in that：The gene has the sequence shown in (a) or (b) Row：

   (a)Seq ID No：Genome nucleotide sequence shown in 1；

   (b) add in the nucleotide sequence shown in (a) and/or substitute and/or lack one or more nucleotides and generate Codified has mutator, allele or the derivative of the protein of rice chloroplast developmental regulation function.
3. A kind of 3. plant expression vector containing gene described in claim 2.
4. 4. plant expression vector according to claim 3, it is characterised in that：The plant expression vector is PCambia1300 expression vectors.
5. A kind of 5. host cell, it is characterised in that：The host cell contains the gene order described in claim 2.
6. 6. host cell according to claim 5, it is characterised in that：The host cell is Bacillus coli cells or agriculture bar Bacterium cell.
7. A kind of 7. method for improveing rice leaf color, it is characterised in that：By the genetic transformation rice cell described in claim 2, then Rice cell after conversion is cultivated into plant.
8. 8. protein described in a kind of claim 1, gene described in claim 2, the plant expression vector of claim 3 or 4, The host cell of claim 5 or 6 is in the regulation and control developmental application of plant chloroplast.
9. 9. application according to claim 8, it is characterised in that：The plant is grass.
10. 10. application according to claim 9, it is characterised in that：The plant is rice.