CN114106129B

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

Info

Publication number: CN114106129B
Application number: CN202111526489.2A
Authority: CN
Inventors: 师家勤; 梁华兵; 王汉中; 王新发; 刘贵华; 詹杰鹏
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-07-29
Anticipated expiration: 2041-12-14
Also published as: CN114106129A

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 particularly relates to application of a rape SWEET15 sugar transporter in improving the yield of rape.

Background

Seed size is an important adaptive feature in plant life history. The seed dispersal, germination, seedling colonization and population distribution pattern are all related to seed weight (size) (Zhu et al, 2012; ZHao et al, 2013). In crop traits, seed size is central and is a central feature of plant life history. Domestication, as a special selection, plays an important role in the development of many crops. Seed weight (size) is one of the important target traits for crop domestication and artificial breeding (Harlan et al, 1973).

The grain weight is one of three constitutive factors of the single-plant yield of the rape, and plays an important role in the formation of the final yield. Although there is a varying degree of negative correlation between the three constitutive factors of oilseed rape yield (whole silique number, kernels per kernel and thousand kernel weight), the correlation coefficient is often not very large (Gupta et al, 2006), which means that the final yield can be increased by increasing the individual yield constitutive factors (e.g. thousand kernel weight). Regional summary data on chinese winter rape in recent 20 years show that in recent years the increase in yield has been attributed primarily to an increase in grain weight, followed by grain number per horn (greedy, et al, 2010). The increase of yield of double-low rape in 2009 from 2000-year was mainly attributed to the increase of the number of crux and thousand seed weight of a single plant (Shuqiying et al, 2010). In 2001 to 2010, the yield of the rape is increased by 11.12%, and the thousand grain weight which is one of yield composition factors is increased maximally and reaches 7.10%, which shows that the increase of the grain weight is the main reason for the improvement of the yield in these years (Wang Jian Sheng et al, 2012; Zhang Fang et al, 2012). Therefore, the thousand seed weight of Chinese rape is increasing year by year in recent years, and the increase of yield is mainly attributed to the increase of thousand seed weight. At present, thousand grain weight of a test variety of the cabbage type rape in China generally does not exceed 4g, and the thousand grain weight can be stabilized to be about 7.5g at most in rape germplasm resources (Li et al.,2014), so that the grain weight improvement has a larger space.

Rape grain weight is a typical quantitative trait, the phenotype is continuously distributed and is easily influenced by environmental conditions, and the phenotype is controlled by a plurality of Quantitative Trait Loci (QTL). With the development of molecular marker technology, one hundred more oilseed rape grain weight QTLs have been currently located using methods of linkage or associative analysis (Bailey-Wilson et al, 2005; quiljada et al, 2006; udallel et al, 2006; Yi bin et al, 2006; radov et al, 2008; Shi et al, 2009; baseunanda et al, 2010a, b; Fan et al, 2010; queen peak et al, 2010; Zhang et al, 2011; Yang et al, 2012; fixed star et al, 2012; Li et al, 2014; Qi et al, 2014). These grain weight QTLs are distributed on all 19 chromosomes of brassica napus, 134 (Zhou et al, 2014) are integrated on the genomic physical map where the sequence information of the linked markers is known, and only a few major QTLs are detected on the a7 (baseunanda 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; fixed star et al, 2012) chromosomes. This strongly suggests that rape grain weight is the quantitative trait controlled by multiple genes, and the genetic basis is very complex.

Although one hundred more grain weight QTLs have been located in oilseed rape, only three major genes have been cloned, including bnaa9.ar F18(Liu et al, 2015), bnaa9.cyp78a9(Shi et al, 2019) and bnaappl 3.c03(Miller et al, 2019). In addition, several genes affecting grain weight have also been identified in oilseed rape using reverse genetics approaches, including BnWRI 1(Liu et al,2010), BnGRF2(Liu et al, 2012), BnDA1(Wang et al, 2017) and bnrbcs (Wu et al, 2017). However, in major crops (mainly rice) and model plants (arabidopsis thaliana) over one hundred genes affecting seed weight (size) have been cloned (lina, 2015), mainly by mutant analysis, followed by map-based cloning.

The applicant finds that the rape SWEET15 sugar transporter (or named as BnaA2.SWEET15 gene) can obviously increase the grain weight and the seed yield by over-expressing in Arabidopsis, and the function of the gene for regulating the seed weight and the final yield is not reported at present. Belongs to a new gene for regulating grain weight and yield, so that the grain weight and yield can be improved by applying the gene to crop breeding.

Disclosure of Invention

The invention aims to provide application of a rape SWEET15 sugar transporter gene in improving rape yield, wherein the rape SWEET15 sugar transporter is shown in SEQ ID NO.2, and the gene can be used for arabidopsis thaliana and brassica napus. BnaA2.SWEET15 increases grain weight and final yield mainly by increasing grain length, as demonstrated by the model plant Arabidopsis.

In order to achieve the purpose, the invention adopts the following technical measures:

the application of rape BnaA2 SWEET15 gene (gene sequence link: http:// cbi. hzau. edu. cn/cgi-bin/bnapus/geneorg ═ ZS11& locus ═ BnaA02G0046800ZS) in improving rape yield includes that BnaA2.SWEET15 gene is over-expressed in plants by utilizing the conventional mode in the field, so that transgenic plants with improved seed length or/and grain width and grain weight can be obtained by screening, and therefore, the application can be used for improving the seed yield of plants.

The plant is Brassica napus or Arabidopsis thaliana.

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

the invention discloses the application of rape gene BnaA2.SWEET15 in the function of regulating and controlling the weight and the yield of seeds for the first time. The transgenic result using arabidopsis as a receptor proves that the seed size, the grain weight and the yield of the overexpression BnaA2.SWEET15 gene are obviously improved compared with the wild type receptor. The present invention therefore proposes that overexpression of BnaA2.SWEET15 can be used to increase grain weight and yield.

Drawings

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

wherein A is the relative expression level of BnaA2 SWEET15 in different transgenic individuals of BnaA2 SWEET15.

B-D are the thousand grain weight, grain number per horn and the phenotype data of the silique length of each positive transgenic line and negative control (Col); the data in the figure are mean ± SD of at least 3 biological replicates, a indicating significant difference at P <0.05, b indicating significant difference at P <0.01, and c indicating significant difference at P < 0.001.

FIG. 2 is a diagram showing the phenotype of BnaA2.SWEET15 gene transgenic positive strain;

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

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

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1: cloning of rape BnaA2.SWEET15 gene coding region

Performing PCR amplification on BnaA2.SWEET15 gene by using a first strand of double 11 cDNA in Brassica napus (Brassica napus L.) as a template, wherein a forward primer of BnaA2.SWEET 15: 5'-ctggtaccatgggcgtcatggtcaatcacc-3', reverse primer: 5'-atgggatcctcaaattcgagacggggcagtc-3', finally obtaining the protein containing the nucleotide sequence shown in SEQ ID NO. 1 and encoding the protein shown in SEQ ID NO. 2.

In the above scheme, KpnI (5 ' -GGTACC-3 ') and BamHI (5 ' -GGATCC-3 ') sites and corresponding protecting bases are added to the 5 ' ends of the forward and reverse primers, respectively, to achieve ligation to the vector.

Example 2: construction of overexpression vectors

The PCR product of the rape BnaA2.SWEET15 obtained by cloning and a plasmid expression vector pCAMBIA2300S (namely, a 35S promoter is added at the position of a multiple cloning site on the basis of a pCAMBIA2300 vector skeleton) are subjected to double enzyme digestion, and the reaction system is as follows:

BamH I: 2 μ L, Kpn I: 2 μ L, 10 × cut smart buffer: 5. mu.L, DNA: 25 μ L, sterilized water: 16 mu L of the solution; a total of 50. mu.L.

React for 2h at 37 ℃.

Then, the double-restriction enzyme plasmid and the PCR product are purified and recovered by a kit according to the instructions, and the concentration of the recovered result is detected by 1% agarose gel.

The connection and transformation of the target gene and the expression plasmid vector comprises the following specific steps:

a. configuration ligation reaction System (10. mu.L)

10×T4 DNA ligase Buffer 1μL

T4 DNA ligase 1μL

DNA fragment (the number of moles of DNA fragment is controlled to 3-10 times that of vector DNA)

ddH ₂ O to 10. mu.L

b, reacting in an incubator at 16 ℃ for more than 12 hours;

c. adding all the above ligation products into 100 μ L DH5 α competent cells, and standing in ice for 20 min;

d.42 ℃ for 90s, and then placing in an ice-water bath for 3 min;

e. adding 400 μ L LB liquid culture medium, culturing at 37 deg.C under shaking at 150rpm/min for 60 min;

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

g. single colonies are picked and cultured in LB solid medium (Kan, 50mg/L) of a slide plate for about 12h, and positive clones are detected by PCR colonies.

Example 3: transformation of Agrobacterium tumefaciens GV3101

Plasmid extraction was performed using the plasmid extraction kit according to the instructions. And detecting whether the target fragment is successfully connected with the expression vector by using double enzyme digestion.

The ice melting method for transforming the agrobacterium comprises the following steps:

(1) adding 2 μ g of purified plasmid into 100 μ L of competent Agrobacterium GV3101, and mixing by gentle shaking;

(2) standing on ice for 5min, immediately placing in liquid nitrogen for 5 min;

(3) water bath at 37 deg.C for 5 min;

(4) adding 800 μ L LB culture medium, shaking and culturing at 28 deg.C in a shaker at 200rpm/min for 1 h;

(5) centrifuging to remove most of supernatant, precipitating, lightly adsorbing with gun, mixing, taking the lower part about 100 μ L of bacterial liquid, and spreading on LB plate containing Rif, Gen and Kan at a concentration of 50 mg/L;

(6) after culturing at 28 ℃ for 48h, resistant colonies can be seen. Selecting a single colony, inoculating the single colony into 2mL LB culture medium (50 mg/L of Rif, Gen and Kan), and carrying out shaking culture at 28 ℃ overnight;

(7) Positive colonies were identified by PCR.

Example 4: agrobacterium-mediated floral dip method for transforming arabidopsis thaliana

Preparing a dip dyeing solution: 5% sucrose + 0.015% surfactant;

YEP culture medium preparation: yeast extract (B): 10g/L, peptone: 10g/L, NaCl: 5 g/L; autoclaving at 121 deg.C for 18 min.

The conversion comprises the following specific steps:

(1) single colonies were picked up in 10mL YEP medium (Kan) ⁺ ：50mg/L，Gen ⁺ ：25mg/L，Rif ⁺ : 25mg/L), and culturing at 28 ℃ and 200rpm/min for 8h with shaking;

(2) taking 500. mu.L of the bacterial solution in (1) and 200ml YEP medium (Kan: 50mg/L, Gen: 25mg/L, Rif: 25mg/L), shaking-culturing at 28 deg.C and 200rpm/min to OD ₆₀₀ ＝1.5-2.0；

(3) And (4) centrifuging the bacterial liquid in the step (2) at 6000rpm/min for 10 min. The precipitate was fully suspended with the conversion dip and the final OD was brought ₆₀₀ Between 1.5 and 2.0;

(4) before transformation, the flowering siliques are cut off;

(5) and (5) dip-dyeing the inflorescences for 30s-1min, culturing in the dark for 24h, and then, converting to normal culture.

(6) One week later, the inflorescences are dip-dyed and transformed once again, the normal culture is carried out until the inflorescences and the fruits blossom and bear the fruits, and the harvested seeds are recorded as T ₀ And (4) generation.

Example 5: screening positive transgenic arabidopsis thaliana:

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

(1) 2.215g MS (Murasing & Skoog basal mediun w/vitamins) is weighed and dissolved in pure water (small amount);

(2) Weighing 24g of sucrose, dissolving, and fixing the volume to 1L;

(3) weighing 8g of Phytagel;

(4) adjusting the pH value to 5.8;

(5) subpackaging and autoclaving (120 deg.C, 20 min).

The method comprises the following steps:

(1) washing with 75% ethanol for 3-5min for 1 time;

(2)ddH ₂ washing for 3 times;

(3) washing with 10% sodium hypochlorite for 3-5 min;

(4)ddH ₂ washing for 3 times;

(5) suspended with a small amount of 0.1% agar.

Resistance screening of transgenic Arabidopsis thaliana and PCR positive identification:

t to be harvested ₀ After the Arabidopsis seeds are disinfected and screened on a resistant plate containing kanamycin (50mg/L) and Meropen (25 mg/L) for bacteriostasis, after 7 to 10 days, the untransformed Arabidopsis seeds do not root and die by yellowing, and the transformed Arabidopsis seedlings can grow normally. After flowering and seed setting, the individual plant is harvested and marked as T ₁ And (4) generation. Will T ₁ T formed by resistant seedlings of arabidopsis thaliana ₁ The seeds are again subjected to resistance screening to obtain T ₂ Resistant seedlings are generated in T2 generations, each strain (15 strains are selected in total and are respectively named as SWEET15-1, 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 and SWEET15-28) selects at least 8 single strains, the whole genome DNA of leaves is extracted, and the plants are subjected to PCR positive identification.

The primers were identified as follows: vector reverse primer: pC2300 s-R: the forward primer BST-Kpn-F of the 5'-gagaaactcgagcttgcatgc-3' target fragment 5'-ctggtaccatgggcgtcatggtcaatcacc-3' amplification system and the method are as follows:

20 μ L reaction system as in Table 1:

TABLE 1 PCR reaction System for Positive plants

PCR reaction procedure:

the amplification reaction procedure was as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and annealing and extension at 72 ℃ for 1min (about 1min for amplification of 1kb according to the length of the fragment), for 34 cycles; extending for 10min at 72 ℃, and storing for later use at 4 ℃. And then carrying out 1% agarose gel electrophoresis detection on the PCR products, wherein the result shows that most of the single plants subjected to resistance screening are positive.

Example 6: real-time fluorescence quantitative RCR detection of expression of transforming gene

15T of the transformation overexpression vector in example 5 ₂ After all the individuals of the generation strains are positively identified, each strain selects mixed leaves of the positive individuals to extract RNA and carries out reverse transcription and real-time fluorescent quantitative PCR to detect the expression condition of the gene.

Arabidopsis beta-actin 1(at2g37620) (Rus et al, 2006) is an internal reference gene. The forward primer of BnaA2 SWEET15 gene is 5'-cagttgatgtcacggtgacg-3', and the reverse primer is 5'-tcaaattcgagacggggcag-3'.

The extraction of Arabidopsis thaliana leaf RNA, the reverse transcription of cDNA first strand and the fluorescent quantitative PCR are all carried out by using a kit according to the instruction manual. The results are shown in FIG. 1, A, and BnaA2.SWEET15 shows higher expression in the transgenic lines.

Example 7: t is a unit of ₂ Phenotypic identification of generation transgenic lines

T used in example 6 ₂ The generation of Arabidopsis seeds, the thousand kernel weight trait examination was performed using SC-G type seed examination and a thousand kernel weight automatic analyzer (ten thousand depth), with a resolution of 1200 pbi. Taking 10 hornberries from each strain, counting the quantity of the hornberries, weighing, and calculating the thousand kernel weight and the number of the hornberries per each strain.

The results are shown in FIGS. 1 and 2: transgenic lines of SWEET15-1, 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 and SWEET15-28 have significantly increased grain weight (B in figure 1) and increased amplitude from 25% -81%. The number of grains per corner of 53% of the lines in these lines was not significantly changed or significantly decreased compared to Col (C in fig. 1), the yield of partial line master sequences was significantly increased compared to Col (D in fig. 1), in addition, the seed was significantly increased mainly in grain length (a in fig. 2), and the grain width of a small number of line seeds was significantly increased (B in fig. 2). It was demonstrated that grain weight is mainly due to increased grain length, and that the increase in grain weight may not be compensated by a decrease in the number of grains per corner, suggesting that this gene can improve yield by increasing grain weight.

Sequence listing

<110> institute of oil crop of academy of agricultural sciences of China

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 (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 (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 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 (Artificial Sequence)

<400> 3

ctggtaccat gggcgtcatg gtcaatcacc 30

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgggatcct caaattcgag acggggcagt c 31

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagaaactcg agcttgcatg c 21

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctggtaccat gggcgtcatg gtcaatcacc 30

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagttgatgt cacggtgacg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaaattcga gacggggcag 20

Claims

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. 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. 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.