CN106434740B - Rape BnbHLH60 gene is improving the application in yield of rape - Google Patents

Abstract

The invention discloses rape BnbHLH60 genes to improve the application in seed weight.The nucleotides sequence of the rape BnbHLH60 gene is classified as shown in SEQ ID NO:1.By the way that the gene is carried out overexpression in arabidopsis, it was found that this transgenic line grain length, grain are wide and grain dramatically increases again, and it is wide that the increase of grain length is greater than grain, and it is mainly realized by increasing cell number, and the influence to every seed number per pod is little, guarantee is provided for the raising of ultimate output, therefore the present invention has good application prospect in the high-yield breeding of crops such as rape.

Description

Rape BnbHLH60 gene is improving the application in yield of rape

Technical field

The invention belongs to plant genetic engineering fields, are more particularly to rape BnbHLH60 gene in improving yield of rape Application.

Background technique

In plant life history, seed size is an important adaptability character.The distribution of seed, sprouting, seedling are fixed Occupy and the Distribution Pattern of population it is all related with seed weight (size) (Zhu et al., 2012；Zhao et al.,2013). In the character of plant, seed size is in Central Position, is a core feature of plant life history.And it tames as a kind of Special selection mode plays an important role in the cultivation of many crops, controls many of Main Agronomic Characters Gene morphs.Seed weight (size) be one of Crop Domestication and the objective trait of biological engineering (Harlan et al., 1973)。

Grain recast is one of three Components of yield of rape, has important role to the formation of ultimate output.Though Different degrees of negative is presented between three Components (complete stool silique number, every seed number per pod and mass of 1000 kernel) of right yield of rape It closes, but its related coefficient is frequently not very big (Gupta et al., 2006), it means that can pass through and improve single yield structure Increase final yield at the factor (such as mass of 1000 kernel).Chinese winter rape region summarized materials is shown within nearly 20 years, in recent years yield Increase be mainly attributed to the increase of grain weight, followed by every seed number per pod (wish sharp rosy clouds etc., 2010).2000-2009 double-low rapeseed The raising of yield is mainly attributed to the increase (Yu Qiying etc., 2010) of single plant knot angle number and mass of 1000 kernel.2001 to 2010 years rapes For yield amplification up to 11.12%, the amplification of one of Yield And Yield Components mass of 1000 kernel is maximum, reaches 7.10%, shows the increase of grain weight Be these years output increased the main reason for (Wang Jiansheng etc., 2012；Virtue etc., 2012).The above results all show in recent years State rape mass of 1000 kernel is in increase trend year by year, and the increase of yield is mainly attributed to the increase of mass of 1000 kernel.It is Chinese for many years sweet Blue type rape novel wheat cultivars mass of 1000 kernel is usually no more than 4g, and mass of 1000 kernel maximum can be stablized on the left side 7.5g in Rape Germplasm Resources Right (Li et al., 2014), therefore grain escheat has very big growth space.

Rape grain is typical quantitative character again, phenotype it is continuously distributed and it is easy influenced by environmental condition, by numerous Quantitative trait locus (quantitative trait loci, QTL) control.With the development of molecular marking technique, utilization is chain Or the method for association analysis located at present the weight of a rape grain more than 100 QTL (Bailey-Wilson et al., 2005； Quijada et al.,2006；Udall et al.,2006；Yi Bin etc., 2006；Radoev et al.,2008；Shi et al.,2009；Basunanda et al.,2010a,b；Fan et al.,2010；Wang Feng etc., 2010；Zhang et al., 2011；Yang et al.,2012；Zhu Heng magnitude, 2012；Li et al.,2014；Qi et al.,2014).These grain weights QTL is distributed on all 19 chromosomes of cabbage type rape, wherein will be integrated into base known to linked marker sequence information Because just there is 134 (Z hou et al., 2014) on group physical map, and only A7 (Basunanda et al., 2010； Fan et al.,2010；Shi et al., 2009) and A9 (Li et al., 2014；Qi et al.,2014；Yang et al.,2012；Zhu Heng magnitude, 2012) a few main effect QTL is detected on chromosome.This pole has strongly suggested rape grain It is the quantitative character of controlled by multiple genes again, hereditary basis is sufficiently complex.

With the successive development of functional genomics, pass through Analysis of Mutants, map based cloning, gene expression analysis and function Dozens of has been cloned in the methods of verifying in staple crops (mainly rice) and model plant (arabidopsis) influences seed The gene (Li Na, 2015) of weight (size).So far, several genes are proved to related with grain weight in rape, and the One rape grain weight QTL is also just come out (Liu et al., 2015) by map based cloning.

This research finds one of gene by some difference expression genes in clone's grain weight main effect QTL region BnbHLH60 is overexpressed in arabidopsis can significantly increase grain weight, which belongs to bHLH transcription factor family, at present function It is unknown.It applies it in crop breeding, grain weight and yield can be improved.

Summary of the invention

The object of the present invention is to provide rape BnbHLH60 genes (gene I/D: 106368187) to improve rape production Application in amount, can be especially useful for arabidopsis and rape.It is confirmed by model plant arabidopsis, BnbHLH60 is by increasing grain Long, the wide final increase grain weight of grain.

To achieve the goals above, the present invention uses following technical measures:

Rape BnbHLH60 gene (gene I/D: 106368187) is improving the application in yield of rape, including the use of ability BnbHLH60 gene is overexpressed by the usual manner in domain in plant, can screen to obtain seed grain weight or/and seed grain length The transgenic plant of raising, therefore can be used for improving the seed production of plant.Schemes described above, it is preferred that the plant Object is rape or arabidopsis.

BnbHLH60 is overexpressed arabidopsis analysis

1) using SC-G type vegetable seeds analysis instrument to T₁And T₂It is scanned and analyzes for the seed of transgenic line, Including grain length, grain is wide and grain again etc..The result shows that many transgenic positive strains are compared with negative control, seed size and Weight increases considerably, and the variation of every seed number per pod is little.

2) to T₁And T₂It is analyzed for the embryonic cell number and size of transgenic line seed, the results showed that transgenosis sun Property strain is compared with control, and blastocyte size variation is little, and embryonic cell number dramatically increases.

Compared with prior art, the invention has the following advantages that

Present invention firstly discloses the basic helix transcription factor gene BnbHLH60 of rape in the function for regulating and controlling seed size Application in energy.Confirm that the overexpression of BnbHLH60 gene increases cotyledon using the transgene result that arabidopsis is receptor With the cell number of plumular axis, and then grain length and grain weight are increased.The present invention is the experimental results showed that the kind of BnbHLH60 gene is careful Born of the same parents' number and grain weight have raising by a relatively large margin compared with wild-type receptor.Therefore the present invention proposes to utilize BnbHLH60's Overexpression increases grain weight, without influencing every seed number per pod, thus eventually for the SOYBEAN IN HIGH-YIELD BREEDING of the crops such as rape.

Detailed description of the invention

Fig. 1 is the verifying of BnbHLH60 recombinant plasmid double digestion.

Swimming lane 1 is BnbHLH60 recombinant plasmid；After 2 are BnbHLH60 recombinant plasmid double digestion.Dotted arrow meaning is mesh Mark genetic fragment.

Fig. 2 is T₀Resistance screening positive seedling.

Arrow meaning is positive seedling.

Fig. 3 is the relative expression levels of gene BnbHLH60 in transgenic line blade.

* indicates significant in the level difference of P < 0.01.

The size comparison schematic diagram of Fig. 4 transgenic plant and wild-type control seeds.

Wherein left figure is wild control, and right figure is the seed of transgenic line.

Transgenic line and wild type control grain the weight associated phenotypic traits of Fig. 5 BnbHLH60.

* indicates significant in the level difference of P < 0.01.

Specific embodiment

Embodiment 1:

The clone of the gene coding region rape BnbHLH60:

Double No. 11 the first chains of cDNA carry out PCR amplification to BnbHLH60 gene as template in, and BnbHLH60 forward direction is drawn Object: 5 '-ACGCGTCGACATGGATCTGACCGGAGCCTTCG-3 ', reverse primer: 5 '-GGGGT ACCTCACAGCTCCATTTTGACCTGG-3 ', it is final to obtain comprising nucleotide sequence shown in SEQ ID NO:1, the nucleotide Amino acid sequence shown in sequential coding SEQ ID NO:2.

In above scheme, in order to realize the connection with carrier, SalI enzyme is added respectively at 5 ' ends of forward and reverse primer Enzyme site (5 '-GTCGAC-3 ') and KpnI restriction enzyme site (5 '-GGTACC-3 ') and corresponding protection base.

Embodiment 2:

The building of over-express vector

Clone is obtained the PCR product of rape BnbHLH60 and plasmid expression vector pD1301S (Huang et al., 2014) double digestion is carried out, reaction system is as follows:

37 DEG C reaction > 12 hours.

Then, double digestion plasmid and PCR product are utilized respectively kit and carry out purification and recovery to specifications, recycle result Its concentration is detected using 1% Ago-Gel.

The connection and conversion of target gene and expression plasmid carrier, the specific steps are as follows:

A. coupled reaction system (10 μ l) is configured

10×T4DNA ligase Buffer 1μl

T4DNA ligase 1μl

DNA fragmentation (3-10 times carrier DNA of the molal quantity control of DNA fragmentation in carrier DNA)

ddH₂O to 10 μ l

B.16 DEG C incubator reacts > 12h；

C. full dose is added in 100 μ l DH5 α competent cells, and 30min is placed in ice；

D.42 after DEG C heating 45s, then 1min is placed in ice；

E. it is added 890 μ l LB liquid mediums, 37 DEG C, 150rpm/min shaken cultivation 60min；

F. the overnight incubation on the LB solid medium of (Kan, 50mg/L) containing kanamycin；

G. picking single bacterium falls within LB solid medium (Kan, the 50mg/L) culture 12 hours or so for drawing plate, while PCR bacterium Fall detection positive colony.

Embodiment 3:

The conversion of Agrobacterium tumefaciems GV3101

The extraction and application plasmid extraction kit operation of plasmid is carried out referring to specification.Target fragment is detected using double digestion Whether with expression vector (Fig. 1) is successfully connected.

Ice melts method conversion Agrobacterium, and method and step is as follows:

(1) the 2 μ g plasmid purified is added in 100 μ l competence Agrobacterium GV3101, gently oscillation mixes；

(2) 10min is placed on ice, is put immediately into liquid nitrogen, 5min；

(3) 28 DEG C of water-bath 5min；

(4) add the LB culture medium of 800 μ l, the 200rpm/min shaken cultivation 4-5h on 28 DEG C of shaking table；

(5) most of supernatant is removed in centrifugation, precipitates, mixing is gently adsorbed with rifle, about 100 μ l bacterium solution of the portion of removing is applied to containing 50m On the LB plate of Rif, Gen and Kan of g/L；

(6) 28 DEG C are cultivated 48 hours, it is seen that resistant bacterium colony is grown.It chooses single colonie and is seeded to 2ml LB culture medium (Rif, G The equal 50mg/L of en, Kan) in, 28 DEG C of shaken cultivations are stayed overnight；

(7) PCR identifies positive bacterium colony.

Embodiment 4:

The flower-dipping method arabidopsis thaliana transformation of mediated by agriculture bacillus:

Dip dyeing liquid for shell configuration :+0.015% surfactant of 5% sucrose；

YEP culture medium is prepared: yeast extract 10g/L

Peptone 10g/L

NaCl 5g/L

High pressure sterilization.

Specific step is as follows for conversion:

1) spot+10ml YEP culture medium (Kan:50mg/L, Gen:25mg/L, Rif:25mg/L) is chosen in inoculation, shakes in 28 DEG C Bed 150rpm/min shaken cultivation (OD₆₀₀=1.5-2.0)；

2) 500 μ l 1) in shaken bacterium solution+200ml YEP culture medium (Kan:50mg/L, Gen:25mg/L, Rif:25mg/ L), in 28 DEG C of shaking table 150rpm/min shaken cultivation (OD₆₀₀=1.5-2.0)；

(3) 6000rpm/min is centrifuged 10min.Precipitating washes (OD600=1.5-2.0) with conversion dip dyeing liquid for shell；

(4) it is cut before conversion colored silique was opened；

(5) inflorescence is disseminated, 30s-1min is disseminated, is cultivated in dark and switch to normally cultivate for 24 hours afterwards.

(6) it is primary to disseminate inflorescence conversion again after a week, to yielding positive results, harvest seed is denoted as T for normal culture₀Generation.

Embodiment 5:

Resistance screening transgenic arabidopsis and acquisition homozygous lines

The configuration of 1/2MS solid medium :+0.8% agar of 1/2MS+24% sucrose (microculture is dedicated) is prepared:

(1) 2.215g MS (Murashing&Skoog basal mediun w/vitamins) is weighed, pure water is added to dissolve (a small amount of)；

(2) 24g sucrose is weighed, dissolves, is settled to 1L；

(3) 8g agar is weighed；

(4) adjusting PH is 5.8；

(5) it dispenses, high pressure sterilization (120 DEG C, 20min).

Arabidopsis seed disinfection, the specific steps are as follows:

(1) 75% ethyl alcohol washes 1 time, 3-5min；

(2)ddH₂O is washed 3 times；

(3) 10% sodium hypochlorite wash one time, 3-5min；

(4)ddH₂O is washed 3 times；

(5) plus 0.1% a small amount of agar suspends.

Resistance screening transgenic arabidopsis and acquisition homozygous lines

By the T of harvest₀After arabidopsis seed disinfection, containing hygromycin (50mg/L) and cephalo (bacteriostasis, 100mg/ L it is screened in resistant panel), after 7-10 days, unconverted arabidopsis seed is not taken root, and yellow is dead, and the arabidopsis converted Seedling being capable of normal growth (Fig. 2).After seedsetting of blooming, single plant system collects seed, is denoted as T₁Generation.By T₁For arabidopsis resistance seedling The T tied₁Seed carries out resistance screening, T again₁The separation that different proportion is had for seed, by segregation ratio close to the strain of 3:1 Single plant collects T₂For seed, T₂For all normal resistance seedlings after seed resistance screening, obtained T₃It is external source for seed The homozygous lines of gene list copy insertion.

Embodiment 6:

The expression of sxemiquantitative RCR detection transformed gene

Two T of overexpression vector₃Homozygous single copy strain (investigating trait phenotypes) is used for semiquantitive PCR to examine Survey the expression of transformed gene.Arabidopsis β-actin1 (at2g37620) and UBQ10 (at4g05320) (Rus et al., It 2006) is reference gene.BnbHLH60 gene forward primer is 5 '-TCATCACCGGCAACTGCAACTG-3 ', and reverse primer is 5’–ATTTTCAGAAACAGCCATCAAACT-3’。

Arabidopsis leaf RNA extraction, the first chain of reverse transcription cDNA, quantitative fluorescent PCR utilize kit referring to specification It carries out.Its result turns in wild type (WT) and empty carrier as shown in figure 3, BnbHLH60 strong expression in transgenic line Change strain (EV) not express substantially, is WT respectively in the expression of transgenic line BnbHLH60line1 and BnbHLH60line2 5 and 11 times.

Embodiment 7:

The PCR of transgenic positive plant is detected

Extract T₀Generation and part T₃It is special using the primer and expression vector of target gene for the blade full genome DNA of resistance seedling Different primer carries out PCR amplification, verifies the positive findings of resistance screening.

Embodiment 8: transgenic line phenotypic evaluation

The character examination of the per generation strain of arabidopsis mass of 1000 kernel uses the species test of SC-G type and mass of 1000 kernel automatic analyzer (Wan Shen) It carries out, the resolution ratio of 1200pbi.Each strain takes five siliques, counts quantity, and weighing calculates mass of 1000 kernel and every seed number per pod.

As a result as shown in Figure 4 and Figure 5: BnbHLH60line1 (present invention or be 35S::BnbHLH60-1) and The transgenic line of BnbHLH60line2 (present invention or be 35S::BnbHLH60-2) significantly increases that grain length, grain be wide and grain Weight, and the increase ratio of grain length is greater than the wide increase ratio (table 1) of grain, illustrates the increase of grain weight mainly by the increase of grain length It is caused.

1 transgenic line grain of table weight, grain length and grain are wide (means standard deviation)

In same row, identical letter indicates not significant in 0.05 level difference.

In addition, every seed number per pod of transgenic line compares that there are no significant with WT difference (Fig. 5), illustrates that the increase of grain weight does not have Have using the reduction of every seed number per pod as compensation, this gene can make again final output increased by increasing grain.

Embodiment 9: cytological observation

Arabidopsis seed impregnates 1 hour in water, embryo and kind skin is removed under simple microscope, by the embryo FAA of removing Fixer fixes 12h, is then transferred in chloral hydrate solution and fixes 24-48h.It is micro- to be finally placed in the inversion of fluorescence number It is observed under mirror (IX71).Using Image J program (Http:// rsb.info.nih.gov/ij/) estimation at least 10A cell The cell number of cell area and the area for obtaining average cell area and cotyledon and radicle, cotyledon and radicle distinguishes root It is obtained according to the area of cotyledon and radicle divided by respective single mean cell area.

The result shows that the cotyledon of transgenic line is compared with the cell size of hypocotyl and wildtype Arabidopsis thaliana without significant Property difference, but the cotyledon of transgenic line and lower plumular axis cell number are significantly greater than wild type seeds (table 2).To Obtain the increase of grain weight mainly due to the increased conclusion of cell number.

The cell size and cell number (means standard deviation) of 2 transgenic line cotyledon of table and hypocotyl

In same row, identical letter indicates not significant in 0.05 level difference.

SEQUENCE LISTING

<110>Inst. of Oil Crops, Chinese Academy of Agriculture

<120>rape BnbHLH60 gene is improving the application in yield of rape

<130>rape BnbHLH60 gene is improving the application in yield of rape

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<170> PatentIn version 3.1

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ctcccgccta cacaagccgt ggagcttctc catttcactg atccctcgcc ctcttcttct 180

caggcggccg tcgccggagt cggtttagac gtctctccgc cgccaccacc gcttcacgct 240

tacggagcat tgacttttcc ttctaacgca gccctcatgg agcgagcagc tcgcttctcg 300

gtgatggcca atgagcagca aaacggaaac gtctccggtg agactaccac gacgagctcg 360

gttccttcca actccgatag agtcaagacc gagcctggtg agacggattc gtctcagcgg 420

ttggagaaca atcgatgtgg caagaggaaa gaactcgaca agaaggtgaa aggctcaacg 480

aagaagaaga gcaaaagctc tgaagagaac gagaagctgc cttatgttca cgttagagct 540

cgccgcggtc aagcaacaga tagccatagc ttagctgaac gagcaagaag agagaagatt 600

aatgcacgaa tgaagctact acaggaactg gtcccaggct gtgataagat tcaaggtact 660

gcgttggtgc tggatgagat cataaaccat gtccagtctt tacaacgtca agtggagatg 720

ctatcaatga gacttgctgc agtaaacccc agaatagact tcaatctcga caccttatta 780

gcctcagaaa atggttcttt aatggatggg agctttaatg gtacagcaat gcagcttgct 840

tggcctcatc aagtcaccga gactgaacaa tcctttcatc accggcaact gcaactgcaa 900

caaccaccac aaccgcaaca atggccttat gatggcttga accagccggc ttggggaaga 960

gaagatgatc aaggtcatgg caatgaacac aacagtttga tggctgtttc tgaaaatttg1020

atggtgggtt ctgctagttt gcacccaaac caggtcaaaa tggagctgtg a 1071

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Cys Arg Glu Pro Pro Gly Leu Glu Ser Leu His Leu Gly Gly Gly Asp

20 25 30

Gly Gly Phe Thr Ala Leu Leu Glu Leu Pro Pro Thr Gln Ala Val Glu

35 40 45

Leu Leu His Phe Thr Asp Pro Ser Pro Ser Ser Ser Gln Ala Ala Val

50 55 60

Ala Gly Val Gly Leu Asp Val Ser Pro Pro Pro Pro Pro Leu His Ala

65 70 75 80

Tyr Gly Ala Leu Thr Phe Pro Ser Asn Ala Ala Leu Met Glu Arg Ala

85 90 95

Ala Arg Phe Ser Val Met Ala Asn Glu Gln Gln Asn Gly Asn Val Ser

100 105 110

Gly Glu Thr Thr Thr Thr Ser Ser Val Pro Ser Asn Ser Asp Arg Val

115 120 125

Lys Thr Glu Pro Gly Glu Thr Asp Ser Ser Gln Arg Leu Glu Asn Asn

130 135 140

Arg Cys Gly Lys Arg Lys Glu Leu Asp Lys Lys Val Lys Gly Ser Thr

145 150 155 160

Lys Lys Lys Ser Lys Ser Ser Glu Glu Asn Glu Lys Leu Pro Tyr Val

165 170 175

His Val Arg Ala Arg Arg Gly Gln Ala Thr Asp Ser His Ser Leu Ala

180 185 190

Glu Arg Ala Arg Arg Glu Lys Ile Asn Ala Arg Met Lys Leu Leu Gln

195 200 205

Glu Leu Val Pro Gly Cys Asp Lys Ile Gln Gly Thr Ala Leu Val Leu

210 215 220

Asp Glu Ile Ile Asn His Val Gln Ser Leu Gln Arg Gln Val Glu Met

225 230 235 240

Leu Ser Met Arg Leu Ala Ala Val Asn Pro Arg Ile Asp Phe Asn Leu

245 250 255

Asp Thr Leu Leu Ala Ser Glu Asn Gly Ser Leu Met Asp Gly Ser Phe

260 265 270

Asn Gly Thr Ala Met Gln Leu Ala Trp Pro His Gln Val Thr Glu Thr

275 280 285

Glu Gln Ser Phe His His Arg Gln Leu Gln Leu Gln Gln Pro Pro Gln

290 295 300

Pro Gln Gln Trp Pro Tyr Asp Gly Leu Asn Gln Pro Ala Trp Gly Arg

305 310 315 320

Glu Asp Asp Gln Gly His Gly Asn Glu His Asn Ser Leu Met Ala Val

325 330 335

Ser Glu Asn Leu Met Val Gly Ser Ala Ser Leu His Pro Asn Gln Val

340 345 350

Lys Met Glu Leu

355

Claims (1)

1. rape BnbHLH60 gene is improving the application in arabidopsis yield.