CN110184280A

CN110184280A - A kind of albumen and the application of the GLW10 gene and its coding controlling rice grain length and mass of 1000 kernel

Info

Publication number: CN110184280A
Application number: CN201910531566.XA
Authority: CN
Inventors: 陈薇兰; 袁华; 钦鹏; 李仕贵; 刘艺; 邓朝杨; 涂斌; 王玉平; 马炳田; 陈郡雯
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2019-08-30
Anticipated expiration: 2039-06-19
Also published as: CN110184280B

Abstract

The invention discloses albumen and the applications of a kind of control rice grain length and the GLW10 gene and its coding of mass of 1000 kernel.The nucleotide sequence of the gene is as shown in SEQ ID NO.1；The amino acid sequence of albumen is as shown in SEQ ID NO.2.Gene of the present invention has the function of positive adjusting and controlling rice grain length and mass of 1000 kernel, can apply to increase rice mass of 1000 kernel and improves rice yield.

Description

It is a kind of control rice grain length and mass of 1000 kernel GLW10 gene and its coding albumen and Using

Technical field

The invention belongs to genetic engineering and Biotechnology in Genetic Breeding fields, and in particular to a kind of control rice grain length and mass of 1000 kernel GLW10 gene and its coding albumen and application.

Background technique

Rice is one of most important cereal crops in China, and using rice as staple food, rice disappears the population in 65% or more the whole nation The amount of expense is extremely huge.Rice is China the first generalized grain crop, and cultivated area, total yield and per unit area yield occupy cereal crops first place, Decisive role is played in ensureing national food security.But in recent years, as China human mortality continues to increase and agricultural workforce The environment such as quantity is constantly reduced and arable area is reduced year by year, global warming influence aggravation, make China's rice industry Development faces many-sided challenge.Therefore, rice yield is further increased, for ensuring China's grain security and agriculture sustainable development Exhibition has highly important strategic importance.

Rice yield belongs to complicated economical character, by panicle number per plant, number of grain per ear, setting percentage and mass of 1000 kernel group At these characters are complicated quantitative characters.Direct compositing factor one of of the mass of 1000 kernel as rice yield, it is by grain shape It is determined with grain-filling degree.Grain shape mainly includes grain length, and grain is wide and thick 3 aspects of grain.Grain shape not only will affect rice production It measures, but also will affect rice quality, especially its exterior quality.Therefore the hereditary basis of grain shape related gene is excavated to rice High yield and high quality breeding be it is very necessary, there is a large amount of grain shape related gene or QTL site to be cloned at present, wherein Grain length main effect QTL site includes: GS3, GL3.1, GS2, OsLG3, OsLG3b/LGY3, TGW3/GL3.3 etc.；The wide main effect QTL of grain Site includes: GW2, GS5, GW8, GW5/GSE5, GL7/GW7, OsOTUB1 etc..

Although at present it has been reported that many grain shape related genes, still go back only the regulatory mechanism research of these genes It is one-sided, the especially interaction between gene, regulated and control network etc. is not clear, and there is an urgent need to further excavate grain shape With mass of 1000 kernel related gene.

Summary of the invention

For above-mentioned deficiency in the prior art, the present invention provides a kind of GLW10 base for controlling rice grain length and mass of 1000 kernel The albumen and application, the gene of cause and its coding have the function of positive adjusting and controlling rice grain length and mass of 1000 kernel.

To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:

A kind of GLW10 gene controlling rice grain length and mass of 1000 kernel, the nucleotide sequence of the gene such as SEQ ID NO.1 institute Show.

Further, the nucleotides sequence of the gene is classified as sequence shown in SEQ ID NO.1 and is substituted, lacks or adds one A or multiple nucleotide, and the nucleotide sequence with identical function albumen can be encoded.

The albumen of above-mentioned GLW10 gene coding, the amino acid sequence of the albumen is as shown in SEQ ID NO.2.

Further, the amino acid sequence of the albumen is that sequence shown in SEQ ID NO.2 is substituted, lacks or adds one Or multiple amino acid, and express the amino acid sequence of identical function.

Plasmid comprising above-mentioned GLW10 gene.

Recombinant expression carrier comprising above-mentioned GLW10 gene.

Transgenic cell line comprising above-mentioned GLW10 gene.

Engineering bacteria comprising above-mentioned GLW10 gene.

Above-mentioned GLW10 gene improves the application in rice yield in improvement rice grain length and mass of 1000 kernel.

The invention has the benefit that

The present invention is research material using the small particle mutant glw10 of EMS mutagenesis in another name for Sichuan Province extensive 498, mixes pond using BSA and is sequenced, In conjunction with MutMap localization method, candidate GLW10 gene is navigated to.It obtains to knock out using CRISPR/Cas9 system editor GLW10 and turn Gene strain, knocking out strain grain length compared with wild type significantly reduces, mass of 1000 kernel decline.And the transgenosis of overexpression GLW10 Plant shows as grain length and mass of 1000 kernel dramatically increases, and illustrates that GLW10 has the function of positive adjusting and controlling rice grain length and mass of 1000 kernel, can answer For increasing rice mass of 1000 kernel and improving rice yield.

Detailed description of the invention

Fig. 1 is GLW10 gene knockout carrier GLW10-BGK03 structural schematic diagram；

Fig. 2 is GLW10 gene overexpression carrier pCAMBIA2300-35S-GLW10-eGFP structural schematic diagram；

Fig. 3 is GLW10 gene knockout target site and knockout plant mutational formats schematic diagram；" " indicates that the position base lacks It loses；

Fig. 4 is GLW10 gene knockout strain grain shape and mass of 1000 kernel species test data；Wherein, " * * " is indicated under 0.01 level Significant difference；

Fig. 5 is GLW10 gene knockout strain grain length and the wide schematic diagram of grain；Scale is 3mm；

Fig. 6 is GLW10 gene overexpression transgenic line quantitative detection；Wherein, " * * " is indicated poor under 0.01 level It is different significant；

Fig. 7 is GLW10 gene overexpression transgenic line grain shape and mass of 1000 kernel species test data；Wherein, " * * " is indicated 0.01 horizontal lower significant difference；

Fig. 8 is GLW10 gene overexpression transgenic line grain length and the wide schematic diagram of grain, scale 3mm.

Specific embodiment

A specific embodiment of the invention is described below, in order to facilitate understanding by those skilled in the art this hair It is bright, it should be apparent that the present invention is not limited to the ranges of specific embodiment, for those skilled in the art, As long as various change is in the spirit and scope of the present invention that the attached claims limit and determine, these variations are aobvious and easy See, all are using the innovation and creation of present inventive concept in the column of protection.

The present invention has been surprisingly found that a granule mutation in EMS (ethylmethane sulfonate) the mutagenesis mutant library in another name for Sichuan Province extensive 498 Glw10 is hybridized building F by body glw10 with wild type another name for Sichuan Province extensive 498₂It is located separately group, granule single plant extreme in group is carried out BSA mixes pond sequencing, using MutMap localization method, navigates to candidate GLW10 gene；It is knocked out using CRISPR/Cas9 system GLW10 has found function of the gene in terms of adjusting and controlling rice grain length and mass of 1000 kernel.

The CRISPR/Cas9 knockout carrier of 1 GLW10 of embodiment constructs

The present invention constructs the knockout carrier of GLW10 using the CRISPR/Cas vector construction kit of hundred lattice companies, specifically Process is as follows:

(1) the targetDesign online tool of the website http://skl.scau.edu.cn/ is utilized, design knocks out target position Point；GLW10 gene nucleotide series (SEQ ID NO.2) is inputted in website, generates the gRNA target sequence T1 of 19bp, the target spot Sequence is as follows:

T1:5 '-GGGTGCTTCCTGTCCAAGC-3 ' (SEQ ID NO.3)

(2) by the above target sequence T1 input hundred lattice company Oligo sequence Photographing On-line websites (http: // 121.41.105.238/index/excrispr), BGK03 carrier is selected, Oligo sequence corresponding with kit is generated, Oligo-F and Oligo-R sequence is as follows:

Oligo-F:5 '-TGTGTGGGGTGCTTCCTGTCCAAGC-3 ' (SEQ ID NO.4)

Oligo-R:5 '-AAACGCTTGGACAGGAAGCACCCCA-3 ' (SEQ ID NO.5)

The above Oligo-F and Oligo-R sequence is synthesized by Sheng Gong biotech firm, then by the Oligo-F of synthesis and Oligo-R primer is dissolved in water to 10 μM.

(3) it is proceeded as follows according to hundred lattice CRISPR/Cas vector construction kit specifications:

Step 1: preparation Oligo dimer

After preparing PCR reaction system (18 μ L Buffer Anneal, 1 μ L Oligo-F, 1 μ L Oligo-R) mixing, use PCR instrument is carried out according to following response procedures: 95 DEG C are heated 3 minutes, are slowly dropped to 20 DEG C later with 0.2 DEG C/sec of speed, cooling Oligo dimer is obtained after renaturation.

Step 2: the above Oligo dimer is constructed to CRISPR/Cas carrier

Reaction system (2 μ L CRISPR/Cas Vector, 1 μ L Oligo dimer, 1 μ L Enzyme are prepared on ice Mix finally adds water to 10 μ L) mix after, react at room temperature 1 hour.

Step 3: the above reaction system is converted into Escherichia coli

Competent escherichia coli cell Trans1-T1 (Beijing Quanshijin Biotechnology Co., Ltd) is taken from -80 DEG C Out, it is placed in ice-water bath and melts；The 5 μ L reaction systems that step 2 is prepared are added, are mixed gently, are incubated for 30 minutes on ice (not shake)；The 42 DEG C of metal baths that are placed on be placed in ice-water bath at once after heat shock 60 seconds, stand 2 minutes；Again to centrifugation 500 μ L nonreactive LB liquid mediums are added in pipe, 37 DEG C of metal baths are recovered 10 minutes；Be placed on shaking table, 200rpm, 37 DEG C of vibrations Culture 1 hour is swung, thallus is made to recover；Proper volume bacterium solution is drawn, it is flat in the LB of resistance containing kanamycin with sterile spreading rod It is gently smoothened on plate, 37 DEG C of inversion overnight incubations.

Step 4: extracting plasmid, obtain GLW10 knockout carrier GLW10-BGK03.

Monoclonal on the above LB plate of picking shake bacterium extract plasmid (according to OMEGA plasmid extraction kit specification into Row), plasmid Song Sheng work biotech firm is sequenced, obtains that correct carrier GLW10-BGK03 is sequenced (see Fig. 1).

The building of 2 GLW10 Overexpression vector of embodiment

It using the reverse transcription reagent box of Takara company, operates to specifications, 500ng total serum IgE is used for reverse transcription and is closed At cDNA.Using the cDNA of the synthesis as the coding region sequence (SEQ ID NO.1) of template amplification GLW10 gene, amplimer F and For R respectively with Kpn I and I restriction enzyme site of BamH (sequence shown in underscore), sequence is as follows:

F:5 '-CGGGGTACCATGGGGTGCTTCCTGTCCA-3’(SEQ ID NO.6)

R:5 '-CGCGGATCCACCTCGCCAGCTATTTTG-3’(SEQ ID NO.7)

The GLW10 coding sequence that the above amplification obtains is connected to plant expression vector using T4 ligase Between I restriction enzyme site of Kpn I and BamH of pCAMBIA2300-35S-eGFP, the Overexpression vector of GLW10 is obtained PCAMBIA2300-35S-GLW10-eGFP (see Fig. 2).

3 GLW10 knockout carrier of embodiment and Overexpression vector rice transformation OryzasativaLcv.Nipponbare

(1) by the GLW10 knockout carrier GLW10-BGK03 built above and Overexpression vector pCAMBIA2300- The plasmid of 35S-GLW10-eGFP converts Agrobacterium EHA105 respectively

The detailed process of conversion method for agrobacterium are as follows: take out EHA105 competent cell, the heart of quickly letting go from -80 DEG C of refrigerators It thaws；Add 1 μ L plasmid in 1 pipe competent cell, places 30 minutes on ice；It is freezed 2 minutes in liquid nitrogen；37 DEG C water-bath 5 minutes, Dissolve cell；The nonreactive LB culture medium of 5 times of volumes is added immediately later, shaking table culture 2-3 is small under conditions of 28 DEG C, 170rpm Shi Hou；It is centrifuged 2 minutes in 7000rpm, settling flux cell is in the LB culture medium that volume is 100 μ L；Then it is coated on containing sharp good fortune On the flat and LB plate of kalamycin resistance, drying, 28 DEG C are cultivated 2-3 days.

(2) by the knockout carrier and Overexpression vector after above-mentioned conversion Agrobacterium, using the method for mediated by agriculture bacillus, Wild rice OryzasativaLcv.Nipponbare kind is converted respectively, obtains the knockout and overexpression transgenic plant of GLW10.

4 GLW10 of embodiment knocks out identification and the phenotypic analysis of plant

Obtain after the GLW10 in embodiment 3 knocks out transgenic plant, blade taken to extract DNA, using detection primer KO-F and KO-R amplification sequencing, determines and knocks out plant mutational formats；The particular sequence of primer KO-F and KO-R are as follows:

KO-F:5’-TGCTTCCCTACTACACACT-3’(SEQ ID NO.8)

KO-R:5’-GGAATGTACTAGCAGCAA-3’(SEQ ID NO.9)

The knockout mutations body strain for obtaining 3 kinds of different mutational formats altogether, is named as KO1, KO2 and KO3 (see Fig. 3).It examines Each strain grain shape phenotype of knockout mutations body is examined, and is compared with wild type OryzasativaLcv.Nipponbare (WT), the result is shown in Fig. 4 and Fig. 5.

Fig. 4 is the rice grain shape detection data of GLW10 knockout mutations body strain and wild type OryzasativaLcv.Nipponbare (WT)；Wherein, A is The grain length comparing result of knockout mutations body strain KO1, KO2, KO3 and wild type OryzasativaLcv.Nipponbare (WT)；B is knockout mutations body strain The wide comparing result of grain of KO1, KO2, KO3 and wild type OryzasativaLcv.Nipponbare (WT)；C is knockout mutations body strain KO1, KO2, KO3 and open country The mass of 1000 kernel comparing result of raw type OryzasativaLcv.Nipponbare (WT).

Fig. 5 is the rice grain schematic diagram of GLW10 knockout mutations body strain and wild type OryzasativaLcv.Nipponbare (WT)；Wherein, A is to strike Except mutant strain KO1, KO2, KO3 and wild type OryzasativaLcv.Nipponbare (WT) rice grain length schematic diagram；B be knockout mutations body strain KO1, KO2, KO3 and the wide schematic diagram of wild type OryzasativaLcv.Nipponbare (WT) rice grain；Illustrated by Fig. 4 and Fig. 5, after knocking out GLW10, the grain length of rice It is decreased obviously with mass of 1000 kernel, being embodied in grain length significantly reduces 10.4%-12%, and mass of 1000 kernel is remarkably decreased 17.9%- 21.5%；And grain is wide then without significant change.

The identification of embodiment 5:GLW10 overexpressing plants and phenotypic analysis

After obtaining the GLW10 overexpressing plants in embodiment 3, divide single plant that blade is taken to extract total serum IgE (public using OMEGA The plant RNA extraction kit of department carries out to specifications).Using the reverse transcription reagent box of Takara company, to specifications 500ng total serum IgE is used for reverse transcription synthesis cDNA by operation.Fluorescence quantitative PCR detection is carried out with primer qGLW10, is drawn with ACTIN Object is as internal reference.Fluorogenic quantitative detection primer sequence is as follows:

QGLW10-F:5 '-GTATGGACCTTACAGCAGTA-3 ' (SEQ ID NO.10)

QGLW10-R:5 '-GAGATGCTGAATACCAAGAAG-3 ' (SEQ ID NO.11)

ACTIN-F:5 '-GACTCTGGTGATGGTGTCAGC-3 ' (SEQ ID NO.12)

ACTIN-R:5 '-GGCTGGAAGAGGACCTCAGG-3 ' (SEQ ID NO.13)

It is separate transgenic the strain OE1, OE2 and OE3 (see Fig. 6) of overexpression to 3 quantitative detections, carries out grain shape and examine It examines, and is compared with wild type OryzasativaLcv.Nipponbare, the result is shown in Fig. 7 and Fig. 8.

Fig. 7 is the rice grain shape testing number of GLW10 overexpressing plants OE1, OE2, OE3 and wild type OryzasativaLcv.Nipponbare (WT) According to；Wherein, A is overexpressing plants OE1, OE2, OE3 and wild type OryzasativaLcv.Nipponbare (WT) seed grain length detection data；B is excessive Express plant OE1, OE2, OE3 and the wide detection data of wild type OryzasativaLcv.Nipponbare (WT) seed grain；C be overexpressing plants OE1, OE2, OE3 and wild type OryzasativaLcv.Nipponbare (WT) seed mass of 1000 kernel detection data.

Fig. 8 is the rice grain schematic diagram of GLW10 overexpressing plants and wild type OryzasativaLcv.Nipponbare (WT)；Wherein, A is excessive Express plant OE1, OE2, OE3 and wild type OryzasativaLcv.Nipponbare (WT) seed grain length detection schematic diagram；B be overexpressing plants OE1, OE2, OE3 and the wide detection schematic diagram of wild type OryzasativaLcv.Nipponbare (WT) seed grain；Illustrated by Fig. 7 and Fig. 8, GLW10 overexpression can mention Grain length and the mass of 1000 kernel for rising rice, are embodied in, and the increased amplitude of grain length is positively correlated with expression, and final mass of 1000 kernel is aobvious It writes and increases 8.5%-12.6%, and the wide no significant difference of grain.

As a result, in conclusion the positive adjusting and controlling rice grain length of GLW10 gene energy and mass of 1000 kernel, can be applied to increase rice grain length And mass of 1000 kernel, improve rice yield.

Sequence table

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Claims

1. a kind of GLW10 gene for controlling rice grain length and mass of 1000 kernel, which is characterized in that the nucleotide sequence of the gene is such as Shown in SEQ ID NO.1.

2. the GLW10 gene of control rice grain length and mass of 1000 kernel according to claim 1, which is characterized in that the gene Nucleotides sequence be classified as sequence shown in SEQ ID NO.1 and be substituted, lack or add one or more nucleotide, and can encode Nucleotide sequence with identical function albumen.

3. the albumen of GLW10 gene coding as claimed in claim 1 or 2, which is characterized in that the amino acid sequence of the albumen is such as Shown in SEQ ID NO.2.

4. albumen according to claim 3, which is characterized in that the amino acid sequence of the albumen is SEQ ID NO.2 institute Show that sequence is substituted, lacks or adds one or more amino acid, and expresses the amino acid sequence of identical function.

5. a kind of plasmid comprising GLW10 gene described in claim 1.

6. a kind of recombinant expression carrier comprising GLW10 gene described in claim 1.

7. a kind of transgenic cell line comprising GLW10 gene described in claim 1.

8. a kind of engineering bacteria comprising GLW10 gene described in claim 1.

9. GLW10 gene as claimed in claim 1 or 2 improves the application in rice yield in improvement rice grain length and mass of 1000 kernel.