CN104031131B - A kind of SIT1 mutain and encoding gene thereof and its application in plant stress tolerance - Google Patents

Abstract

The invention discloses a kind of SIT1 mutain and encoding gene thereof and its application in plant stress tolerance。The invention provides a kind of albumen, be following (a) or (b): the protein that (a) aminoacid sequence shown in sequence in sequence table 3 forms；(b) by the aminoacid sequence shown in sequence in sequence table 3 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 3 derives relevant to plant stress tolerance。The experiment proves that, present invention discover that Oryza sativa L. receptoroid kinase mutant gene SIT1^KE, it imports in plant can improve plant salt endurance, and higher than the salt tolerance of wild type SIT1 gene。Prove that this mutant gene has salt tolerance, for improving, crops salt-resistance provides important theoretical foundation and using value。

Description

A kind of SIT1 mutain and encoding gene thereof and its application in plant stress tolerance

Technical field

The present invention relates to biological technical field, particularly relate to a kind of SIT1 mutain and encoding gene thereof and its application in plant stress tolerance。

Background technology

Salt stress is the topmost abiotic factor affecting crop yield at present in the world。According to the incomplete statistics of FAO (Food and Agriculture Organization of the United Nation), China's salt-soda soil area accounts for more than the 20% of total cultivated area, and owing to irrigation method is improper and the reason such as scarcity of fresh water resources, this ratio is also in continuous growth。Research crop tolerance to salt mechanism, for increasing crops planting area, improves grain yield and has very important significance。

Plant is when suffering extraneous salt stress, plant can make a series of stress, it is concentrated mainly on ion channel and the gene such as regulator and transcription factor thereof about this report on the one hand at present, such as Zhu Jiankang teaches classical SOS (SaltOverlySensitive) system that laboratory is reported, substantially by three main ingredient SOS1, SOS2, SOS3 are constituted, this system outer row regulating plant Na ion by phosphorylation。Also has the gene family transport protein HKT1 of another kind of classics, oneth HKT albumen is cloned out in Semen Tritici aestivi and finds that this gene can have selective affine sodium ion and potassium ion, but AtHKT1 is but not as Semen Tritici aestivi HKT and equally has higher ion selectivity in arabidopsis, but in participating in adjustment arabidopsis sodium ion equilibrium process on the ground and under ground portion, the OsHKT2.1 identified in Oryza sativa L. is then that the absorption that have adjusted the sodium ion when low potassium in root plays a role。And scientists has identified many all kinds of transcription factor relevant with salt stress in transcription factor, such as some zinc finger proteins, MYB, AP2/ERF, WRKY and NAC class transcription factor be all report several big class transcription factor regulating salt response much more relatively, such as Zat12, ZFP179, ZFP182, the genes such as SNAC1, SNAC2, SERF1。Also having only a few is the kinase whose such as OsSIK1 of the receptoroid about the responsible signal sensing being positioned at cell surface about the report of plant salt endurance, Srlk, AtLecRK2, salt stress signal is through a series of intermediate transfer process, induction downstream stress response gene expression, makes plant produce resistance to reactant salt。Receptor-like kinase is one of gene family the hugest in plant, has become a study hotspot in botany field about the research of this genoid。Receptor-like kinase albumen is mainly made up of three parts: the ectodomain of various structures, membrane-spanning domain and conservative intracellular kinase domain。Difference according to receptor-like kinase ectodomain, receptor-like kinase family is divided into multiple subfamily。After before more than 20 years, first receptor-like kinase is found in Semen Maydis, increasing receptoroid kinases is cloned out, and be proved to take part in from growth and development of plants to participating in Response to stress, the not affine process of selfing such as plant, the reception of hormone signal and transmission, the adjustment of growth promoter, the various aspects of the growth and development of plants such as disease resistance response。

Existing result shows that receptoroid kinases probably sensing molecule as the salt stress signal of cell surface plays a role。Therefore, the effect in plant salt tolerance reacts of the research receptor-like kinase, is highly important for illustrating the Salt-resistance mechanism of plant。The discovery of this gene and point mutation form thereof and the research of its function is provided new clue for the mechanism setting forth plant salt endurance and genetic engineering breeding aspect provides new material and using value。

Summary of the invention

It is an object of the present invention to provide a kind of SIT1 mutain and encoding gene thereof。

The SIT1 mutain of the present invention, is following (a) or (b):

A protein that () aminoacid sequence shown in sequence in sequence table 3 forms；

(b) by the aminoacid sequence shown in sequence in sequence table 3 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 3 derives relevant to plant stress tolerance。

The replacement of above-mentioned one or several amino acid residue of process and/or disappearance and/or be added to less than the replacement of 10 amino acid residues and/or disappearance and/or interpolation。

The DNA molecular encoding above-mentioned albumen is also the scope of protection of the invention。

Above-mentioned DNA molecular is the DNA molecular of any one in following (1)-(3):

(1) coding region is the DNA molecular shown in sequence 2 in sequence table；

(2) DNA molecular of albumen relevant to the hybridization of DNA sequence that (1) limits and coding and plant stress tolerance under strict conditions；

(3) DNA sequence limited with (1) at least has 70%, at least has 75%, at least has 80%, at least has 85%, at least has 90%, at least has 95%, at least has 96%, at least has 97%, at least has 98% or at least have 99% homology and the DNA molecular of coding and plant stress tolerance correlative protein。

Above-mentioned stringent condition is as follows: 50 DEG C, at 7% sodium lauryl sulphate (SDS), 0.5MNaPO₄Hybridize with in the mixed solution of 1mMEDTA, at 50 DEG C, 2 × SSC, 0.1%SDS rinses；

Also it is the scope of protection of the invention containing the recombinant vector of above-mentioned DNA molecular, expression cassette, transgenic cell line or recombinant bacterium。

Above-mentioned recombinant vector is inserted in expression vector by above-mentioned DNA molecular, obtains expressing the recombinant vector of above-mentioned albumen。

In an embodiment of the present invention, expression vector is pCAMBIA1300-35S::MH carrier, and recombinant vector is that the nucleotide shown in sequence in sequence table 2 is inserted the carrier obtained between XbaI and the BglII double enzyme site of pCAMBIA1300-35S::MH carrier。

PCAMBIA1300-35S::MH carrier is that the DNA molecular shown in sequence 5 is inserted the carrier that BamHI and the EcoRI restriction enzyme site of pCAMBIA1300 carrier obtains。

The primer pair expanding above-mentioned DNA molecular total length or its any fragment is also the scope of protection of the invention。

The application in regulation and control plant stress tolerance of above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium is also the scope of protection of the invention。

In above-mentioned application, described resistance of reverse is salt tolerance；

Described regulation and control plant stress tolerance is for improving plant stress tolerance；

Described plant is dicotyledon or monocotyledon。

It is a further object to provide a kind of method cultivating transgenic plant。

Method provided by the invention, for the DNA molecular of above-mentioned albumen is imported purpose plant, it is thus achieved that transgenic plant, the resistance of reverse of described transgenic plant is higher than described purpose plant。

In said method, described resistance of reverse is salt tolerance；

Described purpose plant is dicotyledon or monocotyledon。

The experiment proves that, present invention discover that Oryza sativa L. receptoroid kinase mutant gene SIT1^KE, it imports in plant can improve plant salt endurance, and higher than the salt tolerance of wild type SIT1 gene。Prove that this mutant gene has salt tolerance, for improving, crops salt-resistance provides important theoretical foundation and using value。

Accompanying drawing explanation

Fig. 1 is the Salt Tolerance Analysis figure of SIT1 arabidopsis overexpression mutant

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method。

Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain。

Embodiment 1, SIT1 mutant gene SIT1^KEAnd the structure of over-express vector

1, the acquisition of SIT1 gene

Extract Japanese fine rice leaf RNA, it is template that reverse transcription obtains cDNA, pcr amplification is carried out with primer 5 ' GCTCTAGATGCGGCGTCCCGAGCTAA3 ' and 5 ' GAAGATCTCGCTCGAGGAATGTCA3 ', obtain the PCR primer of 2034bp, through order-checking, there is the nucleotide shown in sequence 1, remove the gene of termination codon for SIT1。

2, SIT1 mutant gene SIT1^KEAcquisition

1) process LAN SIT1 gene recombined vector

PCAMBIA1300-35S::MH carrier is that the DNA molecular shown in sequence 5 is inserted the carrier that BamHI and the EcoRI restriction enzyme site of pCAMBIA1300 carrier obtains。

Using cDNA as template, pcr amplification is carried out with 5 ' GCTCTAGATGCGGCGTCCCGAGCTAA3 ' and 5 ' GAAGATCTCGCTCGAGGAATGTCA3 ', obtaining the PCR primer of 2034bp, through order-checking, this PCR primer has sequence 1 from the 1st-2022 nucleotide of 5 ' ends。

By the above-mentioned PCR primer of XbaI and BglII double digestion, the digestion products obtained connects with the pCAMBIA1300-35S::MH carrier through same enzyme action, obtains recombinant vector pCAMBIA1300-35S::SIT1-7M6H, for process LAN SIT1 gene recombined vector。

Through order-checking, this carrier is that from the 1st-2022 nucleotide of 5 ' ends, sequence 1 is inserted the carrier obtained between XbaI and the BglII restriction enzyme site of pCAMBIA1300-35S::MH carrier。

2) SIT1 kinases point mutation carrier

Sequence 1 is the SIT1 of termination codon from the 1st-2022 nucleotide of 5 ' ends。

Gateway method is utilized to be connected into pENTR this fragment^TM/ SD/D-TOPO carrier, obtains recombinant vector pENTR^TM/SD/D-TOPO-SIT1。

Design SIT1^KEPoint mutation primer, with reference to Transgene point mutation kit method recombinant vector pENTR^TM/ SD/D-TOPO-SIT1 produces containing SIT1^KERecombinant vector。

SIT1^KEPoint mutation primer is as follows:

5'GTGGAGATTGCAGTGGAGAAGGTATCCCACG3' and

5'CCACTGCAATCTCCACTCGGGATACCAGTA3'

With XbaI and BglII double digestion containing SIT1^KERecombinant vector, reclaim 2034bp fragment, connect with the carrier pCAMBIA1300-35S::MH through same enzyme action, obtain pCAMBIA1300-35S::SIT1^KE-7M6H。Through order-checking, this carrier is that the nucleotide shown in sequence in sequence table 2 is inserted the carrier obtained between XbaI and the BglII double enzyme site of pCAMBIA1300-35S::MH carrier。

In sequence table, the nucleotide shown in sequence 2 is SIT1 mutant gene SIT1^KE, the Protein S IT1 of this gene code^KEAminoacid sequence be sequence 3 in sequence table。

In sequence table, the nucleotide shown in sequence 1 is SIT1 gene, and the aminoacid sequence of the Protein S IT1 of this gene code is sequence 4 in sequence table。

The sequence 1 of SIT1 gene sports GAG from 5 ' end 1156-1158 position AAG, obtains the SIT1 shown in sequence 2^KE；

The sequence 4 of SIT1 albumen is glutamic acid from the 386th lysine mutation of N ' end, obtains the SIT1 shown in sequence 3^KE。

Embodiment 2, mutant gene SIT1^KEApplication in improving plant stress tolerance

1, SIT1 is turned^KEThe structure of arabidopsis

The recombiant plasmid pCAMBIA1300-35S::SIT1 that embodiment 1 is obtained^KE-7M6H converts Agrobacterium GV3101, and (extracting plasmid order-checking to identify, plasmid is pCAMBIA1300-35S::SIT1 to obtain the GV3101 containing recombiant plasmid^KE-7M6H)。

The genetic transformation of arabidopsis: the deployed conversion medium containing Agrobacterium is poured in sizeable beaker or wide-mouth Cans, will cut arabidopsis (the kind col-0 of arabidopsis of angle fruit and open bud in advance, hereinafter referred to as wildtype Arabidopsis thaliana) the whole aerial parts of recipient plant is all immersed in conversion medium, soaks 5-6min；Take out Arabidopsis plant, by stem, leaf, the unnecessary globule such as take and shake off gently, couch on smooth desktop or stool, it is placed in clean plastic casing Deng globule substantially dry, still the posture couched is maintained, and cover lucifuge insulation, renewal cultivation 16-36h in 22 DEG C of illumination cultivation rooms with black sack；Throw off black bag, arabidopsis is vertically placed again, normally cultivate in 22 DEG C of illumination cultivation rooms；After general growth 2-3 week, angle fruit majority is full, now waters less and nutritional solution as far as possible, promotes seed maturity；After seed maturity, being put in 1.5mlEp pipe, add proper amount of dry drying prescription silica gel, short-term preservation under room temperature, best dry seed of long-term preservation is placed in-70 DEG C of refrigerators, obtains T1 for turning SIT1^KEArabidopsis seed。

Extract T1 for turning SIT1^KEArabidopsis leaf genomic DNA, carries out pcr amplification with 5 '-ATGCGGCGTCCCGAGCTAA-3 ' and 5 '-CGCTCGAGGAATGTCA-3 ', and what obtain 2022bp turns SIT1 for positive T1 generation^KEArabidopsis。

In sowing positive T1 generation, turns SIT1^KEArabidopsis goes down to posterity, until obtaining T3 generation to turn SIT1^KEArabidopsis。T3 utilizes c-MYC antibody (sigma) to detect SIT1 for pure and mild plant by westernblot^KEThe expression of-MYC fusion protein。

Adopting same method to proceed in arabidopsis by pCAMBIA1300-35S::SIT1-7M6H, cultivating until obtaining T3 generation to turn SIT1 arabidopsis。

2、SIT1^KEApplication in improving Salt Resistance of Rice

In T3 generation, is turned SIT1^KEArabidopsis strain 4#, 9# cultivate in the 1/2MS culture medium containing 100mMNaCl, turn SIT1 arabidopsis for comparison with wildtype Arabidopsis thaliana Col and T3 generation。Experiment repeats 3 times, results averaged。

Result is as it is shown in figure 1, A figure is phenotypic map, and B figure is survival rate cartogram, and wherein, SIT1 turns SIT1 arabidopsis, SIT1 in T3 generation^KE9 and SIT1^KE4 turn SIT1 for T3 generation^KEArabidopsis strain, col are wildtype Arabidopsis thaliana；It can be seen that

Under normal operation, each strain is without significant difference；

Under 100mMNaCl coerces, compared with turning SIT1 arabidopsis with wildtype Arabidopsis thaliana and T3 generation, point mutation SIT1^DAIn T3 generation, turns SIT1^KEArabidopsis SIT1^KE9 and SIT1^KE4 all show obvious salt tolerant resistance, turn SIT1^KEThe survival rate (being more than 95%) of arabidopsis, higher than wild-type plant (about 85%), is significantly larger than T3 for turning SIT1 arabidopsis SIT1。

The above results shows, SIT1^KEPlant salt can be improved, in plant salt endurance, play Pasitive Regulation Effect of Genseng。

Claims (7)

1. an albumen, the protein that the aminoacid sequence shown in sequence in sequence table 3 forms。

2. the DNA molecular of albumen described in coding claim 1。

3. DNA molecular as claimed in claim 2, it is characterised in that: described DNA molecular is coding region is the DNA molecular shown in sequence 2 in sequence table。

4. contain the recombinant vector of DNA molecular described in Claims 2 or 3, expression cassette or recombinant bacterium。

5. recombinant vector as claimed in claim 4, it is characterised in that:

Described recombinant vector, for being inserted in expression vector by DNA molecular described in Claims 2 or 3, obtains expressing the recombinant vector of albumen described in claim 1。

6. DNA molecular described in albumen, Claims 2 or 3 described in claim 1 or the application in regulation and control plant stress tolerance of recombinant vector, expression cassette or recombinant bacterium described in claim 4；Described resistance of reverse is salt tolerance；

Described regulation and control plant stress tolerance is for improving plant stress tolerance；

Described plant is dicotyledon。

7. the method cultivating transgenic plant, for importing purpose plant by the DNA molecular of albumen described in coding claim 1, it is thus achieved that transgenic plant, the resistance of reverse of described transgenic plant is higher than described purpose plant；Described resistance of reverse is salt tolerance；Described purpose plant is dicotyledon。