CN114106129B - Application of rape SWEET15 sugar transporter gene in improving rape yield - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a rape SWEET15 sugar transporter gene in improving the yield of rape. The rapeBnaA2.SWEET15The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the coding protein is shown as SEQ ID NO. 2. The gene is over-expressed in Arabidopsis thaliana, and the grain length and grain weight of all transgenic lines are obviously increased, while the grain width and seed yield of partial transgenic lines are also obviously increased, so the invention has good application prospect in high-yield breeding of rape and other crops.

Description

Application of rape SWEET15 sugar transporter gene in improving rape yield

technical field

The invention belongs to the field of plant genetic engineering, and more particularly relates to the application of rapeseed SWEET15 sugar transporter in improving rapeseed yield.

Background technique

Seed size is an important adaptive trait in plant life history. Seed dispersal, germination, seedling settlement, and population distribution patterns are all related to seed weight (size) (Zhu et al., 2012; Zhao et al., 2013). Among crop traits, seed size is central and a central feature of plant life history. As a special selection method, domestication plays an important role in the formation of many crops. Seed weight (size) is one of the important target traits in crop domestication and artificial breeding (Harlan et al., 1973).

Grain weight, as one of the three constituent factors of rapeseed yield, plays an important role in the formation of final yield. Although the three components of rapeseed yield (the number of whole plant pods, the number of pods per pod, and the thousand-seed weight) have different degrees of negative correlation, the correlation coefficients are often not very large (Gupta et al., 2006), which means It is possible to increase the final yield by increasing individual yield components such as thousand kernel weight. The regional summary data of winter rapeseed in China in the past 20 years shows that the increase in yield in recent years is mainly due to the increase in grain weight, followed by the number of grains per horn (Zhu Lixia et al., 2010). The increase in canola yield from 2000 to 2009 was mainly attributed to the increase in the number of knots per plant and the thousand-grain weight (Yu Qiying et al., 2010). From 2001 to 2010, rapeseed yield increased by 11.12%, and one thousand grain weight, one of the yield components, increased the most, reaching 7.10%, indicating that the increase in grain weight was the main reason for the increase in yield in these years (Wang Jiansheng et al., 2012; Zhang Fang et al., 2012) . Therefore, in recent years, the thousand-kernel weight of rapeseed in China has been increasing year by year, and the increase in yield is mainly due to the increase in thousand-kernel weight. At present, the 1000-grain weight of regional test varieties of Chinese cabbage type rapeseed is generally not more than 4g, while the maximum 1000-grain weight of rapeseed germplasm resources can be stabilized at about 7.5g (Li et al., 2014), so there is still more room for improvement of grain weight.

Grain weight of rapeseed is a typical quantitative trait, and its phenotypes are distributed continuously and easily affected by environmental conditions. It is controlled by numerous quantitative trait loci (QTL). With the development of molecular marker technology, more than 100 QTLs of rapeseed weight have been located by linkage or association analysis (Bailey-Wilson et al., 2005; Quijada et al., 2006; Udall et al., 2006; Yi Bin et al., 2006; Radoev et al., 2008; Shi et al., 2009; Basunanda et al., 2010a,b; Fan et al., 2010; Wang Feng et al., 2010; Zhang et al., 2011; Yang et al., 2012; Zhu Hengxing et al., 2012; Li et al., 2014; Qi et al., 2014). These grain weight QTLs are distributed on all 19 chromosomes of Brassica napus, and 134 of them have been integrated into the physical genome map for which the linked marker sequence information is known (Zhou et al., 2014), and only in 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 Hengxing et al., 2012) A few major QTLs were detected on chromosomes. This strongly indicates that rapeseed grain weight is a quantitative trait controlled by multiple genes, and the genetic basis is very complex.

Although more than one hundred grain weight QTLs have been mapped in rapeseed, only three major genes have been cloned, including BnaA9.AR F18 (Liu et al., 2015), BnaA9.CYP78A9 (Shi et al., 2019) and BnaUPL3.C03 (Miller et al., 2019). In addition, several genes affecting grain weight were identified in rapeseed by reverse genetics, including BnWRI 1 (Liu et al, 2010), BnGRF2 (Liu et al., 2012), BnDA1 (Wang et al. , 2017) and BnRBC S (Wu et al., 2017). However, more than one hundred genes affecting seed weight (size) have been cloned in major crops (mainly rice) and model plants (Arabidopsis thaliana) (Li Na, 2015), mainly by mutant analysis, followed by Site cloning.

The applicant found that the rapeseed SWEET15 sugar transporter (the present invention or called BnaA2.SWEET15 gene) can significantly increase grain weight and seed yield by over-expression in Arabidopsis thaliana. At present, the function of this gene to regulate seed weight and final yield has not yet been found. report. It belongs to a new gene that regulates grain weight and yield, so it can be applied to crop breeding to improve grain weight and yield.

SUMMARY OF THE INVENTION

The object of the present invention is to provide the application of rape SWEET15 sugar transporter gene in improving rape yield, the rape SWEET15 sugar transporter is shown in SEQ ID NO.2, and the gene of the present invention can be used for Arabidopsis and Brassica napus rape. It was confirmed by the model plant Arabidopsis that BnaA2.SWEET15 increased grain weight and final yield mainly by increasing grain length.

In order to achieve the above object, the present invention adopts the following technical measures:

Application of rape BnaA2.SWEET15 gene (gene sequence link: http://cbi.hzau.edu.cn/cgi-bin/bnapus/gene?org=ZS11&locus=BnaA02G0046800ZS) in improving rape yield, including the use of conventional methods in the field In this way, the BnaA2.SWEET15 gene is overexpressed in plants, and transgenic plants with increased seed grain length or/and grain width and grain weight can be obtained by screening, and thus can be used to improve the seed yield of plants.

The above-mentioned plants are Brassica napus or Arabidopsis thaliana.

Compared with the prior art, the present invention has the following advantages:

The invention discloses for the first time the application of the rape gene BnaA2.SWEET15 in regulating the function of seed weight and yield. The transgenic results using Arabidopsis thaliana as receptors confirmed that the seed size, grain weight and yield of overexpressing BnaA2.SWEET15 gene were significantly improved compared with wild-type receptors. The present invention therefore proposes that overexpression of BnaA2.SWEET15 can be used to increase grain weight and yield.

Description of drawings

Figure 1 is a schematic diagram of the phenotype of the BnaA2.SWEET15 gene transgenic positive strain;

Wherein, A is the relative expression level of BnaA2.SWEET15 in transgenic individual plants with different BnaA2.SWEET15.

B-D are the 1000-grain weight of each positive transgenic line and the negative control (Col), the number of grains per silique, and the phenotypic data of silique length; the data in the figure are the mean ± SD of at least 3 biological replicates, and a represents at P<0.05 Level difference is significant, b means significant difference at P<0.01 level, c means significant difference at P<0.001 level.

Figure 2 is a schematic diagram of the phenotype of the BnaA2.SWEET15 gene transgenic positive strain;

Among them, A is the grain length of each positive transgenic line and negative control (Col). The data in the figure are the mean ± SD of at least 3 biological replicates, and c indicates a significant difference at the P<0.001 level;

B is the grain width of each positive transgenic line and negative control (Col), the data in the figure are the mean ± SD of at least 3 biological replicates, a means significant difference at P<0.05 level, b means difference at P<0.01 level Significant, c means significant difference at P<0.001 level.

Detailed ways

The technical solutions of the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials, unless otherwise specified, are all derived from commercial channels.

Example 1: Cloning of the coding region of the rape BnaA2.SWEET15 gene

The BnaA2.SWEET 15 gene was amplified by PCR using the first strand of Shuang 11 cDNA in Brassica napus L. as a template, BnaA2.SWEET15 forward primer: 5'-ctggtaccatgggcgtcatggtcaatcacc-3', reverse primer : 5'-atgggatcctcaaattcgagacggggcagtc-3', and finally obtain a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO: 1, encoding the protein shown in SEQ ID NO: 2.

In the above scheme, in order to realize the connection with the vector, a KpnI restriction site (5'-GGTACC-3') and a BamHI restriction site (5'-GGATCC) were added to the 5' ends of the forward and reverse primers, respectively. -3') and corresponding protected bases.

Example 2: Construction of overexpression vector

The PCR product of the cloned rape BnaA2.SWEET15 and the plasmid expression vector pCAMBIA2300S (that is, on the basis of the pCAMBIA2300 vector skeleton, the 35S promoter was added at its multi-cloning site) were all subjected to double restriction digestion, and the reaction system was as follows:

BamHI: 2 μL, KpnI: 2 μL, 10*cut smart buffer: 5 μL, DNA: 25 μL, sterilized water: 16 μL; total 50 μL.

37 ℃ reaction 2h.

Then, the double-enzyme-digested plasmid and the PCR product were purified and recovered using a kit according to the instructions, respectively, and the concentration of the recovery results was detected by 1% agarose gel.

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

a. Configure the ligation reaction system (10 μL)

10×T4 DNA ligase Buffer 1μL

T4 DNA ligase 1μL

DNA fragments (the number of moles of DNA fragments is controlled at 3-10 times the carrier DNA)

ddH ₂ O to 10 μL

b.16℃ incubator reaction>12h;

c. All the above ligation products were added to 100 μL of DH5α competent cells and placed in ice for 20 min;

d. After heating at 42°C for 90s, place in an ice-water bath for 3min;

e. Add 400μL of LB liquid medium, 37°C, 150rpm/min shaking culture for 60min;

f. Culture overnight on LB solid medium containing kanamycin (Kan, 50 mg/L);

g. Pick a single colony and cultivate it in LB solid medium (Kan, 50 mg/L) on the plate for about 12 hours, and at the same time, PCR colonies detect positive clones.

Example 3: Transformation of Agrobacterium tumefaciens GV3101

Plasmid extraction was performed using the plasmid extraction kit according to the instructions. Double-enzyme digestion was used to detect whether the target fragment was successfully ligated with the expression vector.

The ice-melting method is used to transform Agrobacterium, and the method steps are as follows:

(1) Add 2 μg of purified plasmid to 100 μL of competent Agrobacterium GV3101, gently shake and mix;

(2) Place on ice for 5 minutes, immediately place in liquid nitrogen for 5 minutes;

(3) 37℃ water bath for 5min;

(4) Add 800 μL of LB medium, and shake for 1 hour on a shaker at 28°C at 200 rpm/min;

(5) Centrifuge to remove most of the supernatant, precipitate, gently absorb and mix with a gun, take about 100 μL of bacterial liquid from the bottom, and apply it to the LB plate containing 50 mg/L Rif, Gen and Kan;

(6) After culturing at 28°C for 48h, resistant colonies can be seen to grow. A single colony was inoculated into 2 mL of LB medium (50 mg/L for Rif, Gen, and Kan), and cultured overnight at 28°C with shaking;

(7) PCR identified positive colonies.

Example 4: Transformation of Arabidopsis thaliana by Agrobacterium-mediated flower dip method

Dyeing solution configuration: 5% sucrose + 0.015% surfactant;

YEP medium preparation: yeast extract: 10g/L, peptone: 10g/L, NaCl: 5g/L; 121°C, 18min autoclaving.

The specific steps of conversion are as follows:

(1) Pick a single colony in 10mL YEP medium (Kan ⁺ : 50mg/L, Gen ⁺ : 25mg/L, Rif ⁺ : 25mg/L), 28°C, 200rpm/min shaking culture for 8h;

(2) Take 500 μL of bacterial liquid in (1) + 200 ml of YEP medium (Kan: 50 mg/L, Gen: 25 mg/L, Rif: 25 mg/L), and shake at 28° C., 200 rpm/min to OD ₆₀₀ =1.5 -2.0;

(3) Centrifuge the bacterial liquid in (2) at 6000 rpm/min for 10 min. The precipitation is fully suspended with the transformation dip solution, and the final OD ₆₀₀ is between 1.5-2.0;

(4) Cut off the flowered siliques before transformation;

(5) Dipping inflorescences for 30s-1min, cultured in the dark for 24h and then transferred to normal culture.

(6) After one week, the dipped inflorescence was transformed once, and it was cultivated normally until flowering and fruiting, and the harvested seeds were recorded as T ₀ generation.

Example 5: Screening for positive transgenic Arabidopsis:

1/2MS solid medium configuration: 1/2MS+24% sucrose+0.8% agar (special for microbial culture):

(1) Weigh 2.215g MS (Murashing & Skoog basal mediun w/vitamins), add pure water to dissolve (a small amount);

(2) Weigh 24g of sucrose, dissolve, and dilute to 1L;

(3) take by weighing 8g Phytagel;

(4) adjust pH to 5.8;

(5) Subpackage and autoclave (120°C, 20min).

Arabidopsis thaliana seed disinfection, the specific steps are as follows:

(1) Wash once with 75% ethanol, 3-5min;

(2) Wash 3 times with ddH ₂ O;

(3) Wash once with 10% sodium hypochlorite, 3-5min;

(4) Wash 3 times with ddH ₂ O;

(5) Add a small amount of 0.1% agar to suspend.

Resistance screening of transgenic Arabidopsis and PCR positive identification:

The harvested T ₀ Arabidopsis seeds were sterilized and screened on resistant plates containing kanamycin (50 mg/L) and Meropen (25 mg/L bacteriostatic effect). After 7-10 days, untransformed Arabidopsis The mustard seeds did not take root and turned yellow and died, while the transformed Arabidopsis seedlings could grow normally. After flowering and seeding, the seeds of a single line were collected and recorded as T ₁ generation. The T ₁ seeds produced by the T ₁ generation Arabidopsis resistant seedlings were screened again for resistance to obtain T ₂ generation resistant seedlings. 1. SWEET15-2, SWEET15-3, SWEET15-4, SWEET15-6, SWEET15-7, SWEET15-8, SWEET15-12, SWEET15-13, SWEET15-16, SWEET15-18, SWEET15-19, SWEET15-20, SWEET15-24, SWEET15-28) select at least 8 individual plants, extract the whole genome DNA of leaves, and then carry out PCR positive identification on these plants.

The identification primers are as follows: Vector reverse primer: pC2300s-R: 5’–gagaaactcgagcttgcatgc-3’ target fragment forward primer BST-Kpn-F: 5’–ctggtaccatgggcgtcatggtcaatcacc-3’ The amplification system and method are as follows:

The 20 μL reaction system is shown in Table 1:

Table 1 PCR reaction system of positive plants

PCR reaction program:

The amplification reaction procedure was as follows: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, annealing and extension at 72 °C for 1 min (adjust the time according to the length of the fragment, about 1 min to amplify 1 kb), 34 cycles; extension at 72 °C for 10 min, Store at 4°C for later use. The above PCR products were then detected by 1% agarose gel electrophoresis, and the results showed that most of the individual strains screened for resistance were positive.

Example 6: Real-time fluorescent quantitative RCR detects the expression of transformed genes

After all the individual plants of the 15 T ₂ generation strains transformed with the overexpression vector in Example 5 were positively identified, the mixed leaves of the positive individual strains were selected from each strain for RNA extraction, reverse transcription and real-time fluorescence quantitative PCR. to detect the expression of the gene.

Arabidopsis β-actin1 (at2g37620) (Rus et al., 2006) was the internal reference gene. The forward primer of BnaA2.SWEET15 gene was 5’–cagttgatgtcacggtgacg-3’, and the reverse primer was 5’–tcaaattcgagacggggcag-3’.

RNA extraction from Arabidopsis thaliana leaves, reverse transcription of the first strand of cDNA, and real-time quantitative PCR were performed using the kit according to the instructions. As a result, as shown in A in Figure 1, BnaA2.SWEET15 was highly expressed in these transgenic lines.

Example 7: Phenotypic identification of T ₂ generation transgenic lines

For the T ₂ generation Arabidopsis thaliana seeds used in Example 6, the 1000-grain weight trait was tested using SC-G type testing and 1000-grain weight automatic analyzer (Wan Shen), with a resolution of 1200 pbi. Take 10 siliques from each strain, count the number, weigh, and calculate the thousand-grain weight and the number of grains per silique.

The results are shown in Figure 1 and Figure 2: SWEET15-1, SWEET15-2, SWEET15-3, SWEET15-4, SWEET15-6, SWEET15-7, SWEET15-8, SWEET15-12, SWEET15-13, SWEET15-16, The grain weight of the transgenic lines of SWEET15-18, SWEET15-19, SWEET15-20, SWEET15-24, and SWEET15-28 was significantly increased (B in Figure 1), and the increase was from 25% to 81%. 53% of these lines showed no significant change or a significant decrease in the number of grains per horn compared to Col (C in Fig. 1), some lines showed a significant increase in main sequence yield compared with Col (D in Fig. 1), and in addition , the main grain length of seeds increased significantly (A in Figure 2), and the grain width of seeds of a few lines increased significantly (B in Figure 2). It shows that the grain weight is mainly caused by the increase of grain length, and the increase of grain weight may not be compensated by the decrease of the number of grains per horn, indicating that this gene can increase the yield by increasing the grain weight.

sequence listing

<110> Oil Crops Research Institute, Chinese Academy of Agricultural Sciences

<120> Application of rape SWEET15 sugar transporter gene in improving rape yield

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 894

<212> DNA

<213> Artificial Sequence

<400> 1

atgggcgtca tggtcaatca ccacttgctc gctatcatct tcggcatctt aggaaacgca 60

atatccttcc ttgtattcct ggctcccgtg ccgacgtttt atagaatata caagaacaaa 120

tcgactgaaa gtttccagtc tctaccgtac caagtgtcac tatttagctg catgctatgg 180

ctctattacg cattgactaa gcaagacgct tttctcctaa ttaccatcaa ctctttcggc 240

tgcgttgtgg agactatcta cattgccatg ttcttcactt acgctaccaa ggagaaaaag 300

atggcggcta ttaagttgtt cttgacgatg aatgttgctt tcttctcgtt gattataatg 360

gttacacatt ttgcggttaa acgccctagc ctccaagtct ctgtcatcgg ctggatttgc 420

gttgctatat ctgtttctgt tttcgctgcc cctctaatga ttgtggctcg tgtgataaag 480

accaagagtg tggagttcat gcccttcacg ctttctttct tcctcactat aagcgctgtt 540

atgtggttcg catatggcgc atttctccac gacatatgca ttgctattcc aaacgtggtg 600

ggattcatac tagggttggt acaaatggtt ttgtatggag tttacagaaa ctcaggggag 660

aaattagata ttgggaaaaa gaataacagt tcatcagaac aacttaagac tattgttgtg 720

atgagtccgt taggtttgtc ggaaatgcac ccagttgatg tcacggtgac ggaaccggtg 780

attccactct cttacactgt tcatcatgaa gatccatcca aaattactaa agaggaggag 840

acgtcaactg aagccgcaca aagccatgtg gagactgccc cgtctcgaat ttga 894

<210> 2

<211> 297

<212> PRT

<213> Artificial Sequence

<400> 2

Met Gly Val Met Val Asn His His Leu Leu Ala Ile Ile Phe Gly Ile

1 5 10 15

Leu Gly Asn Ala Ile Ser Phe Leu Val Phe Leu Ala Pro Val Pro Thr

20 25 30

Phe Tyr Arg Ile Tyr Lys Asn Lys Ser Thr Glu Ser Phe Gln Ser Leu

35 40 45

Pro Tyr Gln Val Ser Leu Phe Ser Cys Met Leu Trp Leu Tyr Tyr Ala

50 55 60

Leu Thr Lys Gln Asp Ala Phe Leu Leu Ile Thr Ile Asn Ser Phe Gly

65 70 75 80

Cys Val Val Glu Thr Ile Tyr Ile Ala Met Phe Phe Thr Tyr Ala Thr

85 90 95

Lys Glu Lys Lys Met Ala Ala Ile Lys Leu Phe Leu Thr Met Asn Val

100 105 110

Ala Phe Phe Ser Leu Ile Ile Met Val Thr His Phe Ala Val Lys Arg

115 120 125

Pro Ser Leu Gln Val Ser Val Ile Gly Trp Ile Cys Val Ala Ile Ser

130 135 140

Val Ser Val Phe Ala Ala Pro Leu Met Ile Val Ala Arg Val Ile Lys

145 150 155 160

Thr Lys Ser Val Glu Phe Met Pro Phe Thr Leu Ser Phe Phe Leu Thr

165 170 175

Ile Ser Ala Val Met Trp Phe Ala Tyr Gly Ala Phe Leu His Asp Ile

180 185 190

Cys Ile Ala Ile Pro Asn Val Val Gly Phe Ile Leu Gly Leu Val Gln

195 200 205

Met Val Leu Tyr Gly Val Tyr Arg Asn Ser Gly Glu Lys Leu Asp Ile

210 215 220

Gly Lys Lys Asn Asn Ser Ser Ser Glu Gln Leu Lys Thr Ile Val Val

225 230 235 240

Met Ser Pro Leu Gly Leu Ser Glu Met His Pro Val Asp Val Thr Val

245 250 255

Thr Glu Pro Val Ile Pro Leu Ser Tyr Thr Val His His His Glu Asp Pro

260 265 270

Ser Lys Ile Thr Lys Glu Glu Glu Thr Ser Thr Glu Ala Ala Gln Ser

275 280 285

His Val Glu Thr Ala Pro Ser Arg Ile

290 295

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 3

ctggtaccat gggcgtcatg gtcaatcacc 30

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 4

atgggatcct caaattcgag acggggcagt c 31

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 5

gagaaactcg agcttgcatg c 21

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 6

ctggtaccat gggcgtcatg gtcaatcacc 30

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

cagttgatgt cacggtgacg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

tcaaattcga gacggggcag 20

Claims (3)

1. The application of the protein shown in SEQ ID NO.2 or the gene for coding the protein in improving the yield of arabidopsis thaliana.
2. 2. The use of claim 1, wherein the increase in yield of Arabidopsis thaliana is achieved by increasing the seed weight of Arabidopsis thaliana by using the protein of SEQ ID No.2 or a gene encoding the protein.
3. 3. The use of claim 1, wherein the protein of SEQ ID No.2 or the gene encoding the protein increases Arabidopsis yield by increasing Arabidopsis seed length and/or seed width.

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