CN102911966A - Application of arabidopsis thaliana At4g31910 gene on aspect of improving rice plant type characters - Google Patents

Abstract

The invention belongs to the technical field of transgenosis and relates to application of an arabidopsis thaliana At4g31910 gene on an aspect of improving rice plant type characters. Particularly rice nipponbare is adopted to serve as a transgenosis receptor, a carrier pCAMBIA1306 with the arabidopsis thaliana gene At4g31910 is used for constructing an overexpression transgenosis plant type of At4g31910, and overexpression of the gene At4g31910 can be used for enabling BR signal output to be weakened to improve a plant type of rice, dwarf the plant type and enable the plant type to easily resist falling.

Description

The application of Arabidopis thaliana At4g31910 gene aspect improvement plant type of rice proterties

Technical field

The invention belongs to field of transgenic technology, be specifically related to the application of Arabidopis thaliana At4g31910 gene aspect improvement plant type of rice proterties.

Background technology

China is large agricultural country, and paddy rice is the main food crop of China.Therefore improving the output of paddy rice, is the target that whole world scientist works hard.The output that improves paddy rice can be by many-sided means, comprise the improvement that realizes Economic Character of Rice by transgenosis means etc.The transgenosis of paddy rice is the relation according to known functional gene and phenotypic character, adopt constitutive expression carrier or tissue specificity expression vector that goal gene is imported target plant, to obtaining the specific economic characters of crop or the good transgenic line of degeneration-resistant proterties.On the basis that does not affect rice fertility and single plant yield, the dwarfing of plant can make paddy rice more resistant to lodging, thereby increases the rice yield on the unit surface.

Rape element sterol (Brassinosteroid, be called for short BR) be the sixth-largest hormone of plant, at pollen, rich content in seed and the tender tissue, it has regulated and control many processes of growth and development of plants, comprise the growth of leaf, the elongation of stem, the differentiation of xylem, male fertile, old and feeble and to resistance [Yang, CJ., the Zhang of biological and abiotic stress, C., Lu, YN, and Wang XL (2011), The mechanisms of Brassinosteroid ' s Action:From Signal Transduction to Plant Development. Molecular Plant 4 (4), 588-600.] [ Shimada, Y., Goda, H., Nakamura, A., Takatsuto, S., Fujioka, S. and Yoshida, S.(2003) Organ-specific Expression of brassinosteroid-biosynthetic genes and distribution of endogenous brassinosteroids in Arabidopsis. Plant Physiology, 131, 287-297.].

In Arabidopis thaliana, after the acyltransferase At4g31910 that acetyl-CoA relies on crosses in Arabidopis thaliana and expresses, can make plant become short and small, the mutant of similar BR content minimizing and signal weakening.Therefore, the gene of seeking Dwarfing Gene has forwarded in the middle of the paddy rice, can improve the output of paddy rice with expectation improvement plant type of rice proterties.

Summary of the invention

The object of the invention is to provide the application of Arabidopis thaliana At4g31910 gene aspect improvement plant type of rice proterties.

Content of the present invention is: adopting paddy rice Japan fine is transgene receptor, uses the carrier pCAMBIA1306 with arabidopsis gene At4g31910, makes up the mistake express transgenic plant of At4g31910 gene.

Among the present invention, with the carrier pCAMBIA1306 of Gene A t4g31910, be that gene with Arabidopis thaliana At4g31910 is inserted between the KpnI in pCAMBIA1306 carrier 35S constitutive promoter downstream and the BamHI restriction enzyme site and obtains.

Among the present invention, the clone of arabidopsis gene At4g31910 obtains its amplification take the cDNA of Arabidopis thaliana as template by PCR.

Among the present invention, the cDNA sequence of arabidopsis gene At4g31910 is as described in the SEQ.ID.NO.1.

Among the present invention, the protein sequence of arabidopsis gene At4g31910 is as described in the SEQ.ID.NO.2.

The present invention includes:

1) Arabidopis thaliana At4g31910The structure of expression vector

Employing pCAMBIA1306 is expression vector, with arabidopsis gene At4g31910Gene is inserted between the KpnI in pCAMBIA1306 carrier 35S constitutive promoter downstream and the BamHI restriction enzyme site (Fig. 1).The gene of Arabidopis thaliana coding acyltransferase At4g31910Full-length cDNA is seen SEQ.ID.NO.1.

2) with gene At4g31910Carrier pCAMBIA1306 rice transformation test and result

Utilizing Japan fine is transgene receptor.With rice paddy seed shell the sterilization after be seeded in evoked callus on the dedifferentiation plant tissue culture media.After callus induction 2-4 week, adopt the method that infects, will contain At4g31910The recombinant plasmid of gene imports Japanese fine callus cell.On the MS substratum that adds the 50mg/L Totomycin, pluck in cultured continuously 3-4 week and select resistant cell line, then the clone of screening is transferred to division culture medium and induced and sprout and take root.Finishing screen is selected independently transformation plant.

Among the present invention, be used for rice transformation At4g31910The albumen (SEQ.ID.NO.2) of gene order (SEQ.ID.NO.1) and translation, the PCR primer of use is:

4g31910-F(KpnI)	AAGGATCCCTTATACAATATGCCCATGTTAAATG
		4g31910-R(BamHI)	AAGGATCCGCAATCAAGGAAATGATTTGAAAAGC

The present invention utilizes arabidopsis gene At4g31910Encoding sequence to have made up constitutive expression carrier rice transformation Japan fine, obtained than the short and small Transgenic Rice material of contrast plant type, improve the plant type shape of paddy rice, improved the lodging resistance of paddy rice, thereby can reach the benefit that increases the rice yield on the unit surface.

Description of drawings

Fig. 1 is the multiple clone site schematic diagram of pCAMBIA1306.

Fig. 2 is transgenosis T1 generation 8 transformants and turning in the unloaded seedling At4g31910The expression amount schematic diagram of gene, wherein, it is fine that Ni represents Japan, and 1306-1 and 1306-2 representative turn 2 unloaded strains, and T1-1 is 8 to T1-8 and turns At4g31910The transformation plant of gene.

Fig. 3 is the phenotype schematic diagram of seedling, and is left for turning the transfer-gen plant of 1306 zero loads, right for turning At4g31910The plant of gene.

Fig. 4 is transgenosis T2 generation 2 strain different plants and turning in the unloaded plant At4g31910The expression amount schematic diagram of gene, wherein, 1306-1 and 1306-2 represent T2 for 2 strains of idle running, and T2 and T6 are that T2 is for turning At4g31910The the 2nd and the 6th strain in the gene strain, wherein each strain is selected 3 seedlings, and submeter is with 1,2, and 3 represent.

Fig. 5 is that the expression amount that 2 the strain different plants contrasts of transgenosis T2 generation turn the BR marker gene in the unloaded seedling changes schematic diagram.

Embodiment

The following examples are to further specify of the present invention, rather than limit the scope of the invention.

Embodiment 1.

The structure of transgene carrier

First by PCR take the cDNA of Arabidopis thaliana Col-0 as the target fragment of template amplification with restriction enzyme site At4g31910The cDNA sequence, employing pCAMBIA1306 is expression vector, with arabidopsis gene At4g31910Be inserted between pCAMBIA1306 carrier 35S constitutive promoter downstream KpnI and the BamHI (Fig. 1).

The Transgenic Rice flow process

1. induce

Peel off outer, the inner glume of seed, the ripe full seed of hanking, the centrifuge tube of the 50ml that packs into.Sterilized water 30ml cleans 4～5 times, 70% alcohol immersion, 2 min, and sterile water wash 3 times is used sterile water wash 4～5 times, 0.15% HgCl again ₂, 100 rpm, 15～20 min.Open at aseptic super clean bench, pour out HgCl ₂, with sterile water wash 4～5 times, be poured on the flat board of sterilization and the filter paper and blot, approximately about 1h.It is inserted (10/25 ml/ bottle) on the N6D solid medium, embryo up or the contact substratum, 28 ℃, secretly cultivate 25～30d.

2. subculture

The callus that observation is induced, the appearance of the energy self falling of generally looking just can peel to carry out succeeding transfer culture.Remove endosperm, plumule, change callus over to new N6D, cultivated 7～10 days.If the callus that peels is larger, can with tweezers it be attenuated in this step.

(1) front cultivation (PH=5.6) turns again the callus of subculture successively that substratum gets final product, and should guarantee that also substratum dries up and callus is dry.Just can infect in general 3～4 days.

(2) cultivation of Agrobacterium is seeded in an amount of Agrobacterium (about 100 μ l) coating on the LB solid medium, and dark 36 h that cultivate of EHA105 and LBA4404 get final product.

(3) infection and altogether cultivation

Suspend: get the little spoon of sterilization scraping Agrobacterium, with spoonful back side with thalline be attached to tube wall clap gently loose, OD ₆₀₀=0.8～1.0.(generally scrape 5～6 spoonfuls of bacterium and get final product, namely see through the bacterium liquid centrifuge tube scale that mays be seen indistinctly, concentration can not be too large, otherwise will dilute).

Infect: the callus of front cultivation is dried in the air at aseptic filter paper, then be concentrated to disposable changing in the bacterium liquid of plate, rotate gently centrifuge tube bacterium liquid is evenly distributed, time of repose approximately 15～20 min (is adjusted according to concentration is different, concentration is large, and the time is just relatively short).

Cultivate altogether: bacterium liquid is poured out, and callus is put approximately 1.5 h at aseptic filter paper, guarantees that bacterium liquid blots, be connected among the 1/2 N6D AS, and 20 ℃, secretly cultivated 2～3 days, see that callus and substratum contact part have mycoderm, just can degerming.Often do not drive incubator in the culturing process altogether, in order to avoid temperature variation is too large, produce moisture film.

3. the removal of Agrobacterium

With pack into the centrifuge tube of 50 ml of the callus of common cultivation, more than 3 times, more limpid to flowing fluid ratio with sterile water wash.Pour out sterilized water, N6D+Cn500 mg/L (or AP500ml/L), 100 rpm, 15-20 min, 2-3 time.Callus is poured on blots on the aseptic filter paper about 2h, depend on the circumstances.The callus of drying is changed among the N6D-AS, do not add Hn, add Cn250 mg/L, secretly cultivate 7～10d by 28 ℃.

4. the screening of callus

Choose the callus of not polluted by Agrobacterium, add Cn250 mg/L and Hn(50 mg/L for the first time), 15～20d.If in current screening, still have callus contaminated, choose and good turn plate screening once.For the second time the same, do not add Cn, add Hn, all callus are all turned once again 15～20d.Select for the third time new callus, with Hn screening, 15～20d.Above-mentioned arrangement is pressed with certain by number of times section, but should guarantee callus more than times at least 45 d that Hn screens, and the callus that newly grows of choosing is for the third time preferably screened 20 d.

5. differentiation

Whole callus of the 4th screening are moved among the MS Hn 50 mg/L, the dark cultivation, pre-differentiation (PH 5.9) 12～15d.Select the good fresh callus (faint yellow) of growing way, move among the MS (PH 6.0), light is cultivated 15～20 d, can see having green bud to grow, and general 15 d change a subculture.If the callus differentiation is relatively good, substratum looks also more pure and fresh, can not change.Choosing grows the above green bud of 1cm, peels off unnecessary callus on every side, cuts off root (can stay approximately 0.5cm length) and moves in the test tube 1/2 MS root culture.

6. the detection of transformation plant

The transformant of survival after the screening is detected candidate's transformant by real-time quantitative PCR, obtain the transformation plant that 8 mistakes are expressed At4g31910.

Endogenous through Real-time PCR Analysis At4g31910Fine and change the seedling of pCAMBIA1306 zero load over to and change in contrast Japan At4g31910Expression amount in the gene plant

Detect a transgenosis T1 of 14 days of seedling in generation with real-time quantitative PCR At4g31910The expression of gene turns the empty carrier plant as contrast so that Japan is warm and fine, uses Actin as confidential reference items (Fig. 2).Find At4g31910High level expression in transfer-gen plant raises multiple and does not wait to thousands of times from hundreds of.

At4g31910The plant type generation considerable change that contrast turns unloaded plant is revealed in transgenic paddy rice T1 representative.Turn At4g31910The paddy rice of gene shows whole plant than the plant height of unloaded seedling short (Fig. 3).

7, T2 is for the detection of BR marker gene level

T2 detects the BR marker gene for seedling by real-time quantitative PCR DWARF, D2, and DWF4Expression amount.

Chosen wherein two transgenic lines that two growth phenotypes are different from contrast, each strain selected 3 plant, and the marker gene of BR signal output is carried out the detection of transcriptional level.At first detected in this 6 strain plant At4g31910Content (Fig. 4).Further find the BR synthase gene DWARF, D2With DWF4Expression raise (Fig. 5) at T2 in for transgenic plant, show that the output of BR signal in these strains has weakened, thereby caused the dwarfing of plant.

SEQUENCE LISTING

＜110〉Fudan University

＜120〉application of Arabidopis thaliana At4g31910 gene aspect improvement plant type of rice proterties

<130>

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1377

<212> DNA

<213>

<400> 1

atgcccatgt taatggcgac acgtatcgat ataatccaaa agcttaatgt atatccaagg 60

tttcaaaacc atgacaagaa gaaactaatc actctctcca atttggaccg tcagtgtcct 120

ttactcatgt actctgtctt cttctacaag aataccacaa ctcgtgactt tgactccgtc 180

ttctccaacc tgaagctcgg gctggaggag actatgtctg tgtggtatcc cgcggcaggg 240

agactgggtt tggacggagg tggctgcaag ctcaacatcc ggtgtaacga tggtggcgca 300

gtcatggtgg aggcggtggc gacaggtgtc aagttgtccg agcttggtga tttgactcag 360

tacaatgagt tttatgagaa tttagtttac aagccttcct tggatggtga tttctctgtg 420

atgcctcttg ttgttgctca ggtgacaaga tttgcatgtg gaggttactc aattggaatc 480

ggtacaagcc actctctatt tgatggaatc tcagcttacg aattcattca cgcgtgggcc 540

tccaactctc acattcacaa caaatccaac agcaagatta ctaataaaaa ggaagatgtg 600

gtcatcaaac cggttcatga tcgacgaaat ctactggtta accgggatgc tgtccgagaa 660

accaatgctg cagccatttg tcatctgtac cagttgatca aacaggcgat gatgacctat 720

caggagcaaa accgtaactt agagttacca gactctggtt ttgtgatcaa aacgttcgag 780

cttaatggcg atgcgataga aagcatgaag aagaaatcac tagaagggtt catgtgctcc 840

tcctttgagt ttcttgctgc tcatttgtgg aaggcaagaa caagggcttt agggttgagg 900

agagacgcca tggtgtgttt acaattcgca gtggacataa ggaaaagaac ggagacaccg 960

ctgccagaag ggttttccgg caacgcatac gtgcttgcct cggtggcatc caccgccaga 1020

gaattacttg aagagctaac actcgagtca atagtcaaca agatcagaga agccaagaaa 1080

tcaattgacc aaggttacat aaactcttac atggaagcac tcggaggtag taatgacgga 1140

aatctccctc ctctcaaaga gctaacccta atctccgact ggacaaaaat gccatttcac 1200

aatgttggct ttggcaacgg cggcgagcca gcggattaca tggccccact gtgtccaccg 1260

gtgccacaag ttgcttattt catgaagaac cctaaagatg ccaaaggtgt tcttgtgagg 1320

attggcttgg acccacgaga tgttaatggc ttttcaaatc atttccttga ttgctaa 1377

<210> 2

<211> 458

<212> PRT

<213>

<400> 2

<400> 2

Met Pro Met Leu Met Ala Thr Arg Ile Asp Ile Ile Gln Lys Leu Asn

1 5 10 15

Val Tyr Pro Arg Phe Gln Asn His Asp Lys Lys Lys Leu Ile Thr Leu

20 25 30

Ser Asn Leu Asp Arg Gln Cys Pro Leu Leu Met Tyr Ser Val Phe Phe

35 40 45

Tyr Lys Asn Thr Thr Thr Arg Asp Phe Asp Ser Val Phe Ser Asn Leu

50 55 60

Lys Leu Gly Leu Glu Glu Thr Met Ser Val Trp Tyr Pro Ala Ala Gly

65 70 75 80

Arg Leu Gly Leu Asp Gly Gly Gly Cys Lys Leu Asn Ile Arg Cys Asn

85 90 95

Asp Gly Gly Ala Val Met Val Glu Ala Val Ala Thr Gly Val Lys Leu

100 105 110

Ser Glu Leu Gly Asp Leu Thr Gln Tyr Asn Glu Phe Tyr Glu Asn Leu

115 120 125

Val Tyr Lys Pro Ser Leu Asp Gly Asp Phe Ser Val Met Pro Leu Val

130 135 140

Val Ala Gln Val Thr Arg Phe Ala Cys Gly Gly Tyr Ser Ile Gly Ile

145 150 155 160

Gly Thr Ser His Ser Leu Phe Asp Gly Ile Ser Ala Tyr Glu Phe Ile

165 170 175

His Ala Trp Ala Ser Asn Ser His Ile His Asn Lys Ser Asn Ser Lys

180 185 190

Ile Thr Asn Lys Lys Glu Asp Val Val Ile Lys Pro Val His Asp Arg

195 200 205

Arg Asn Leu Leu Val Asn Arg Asp Ala Val Arg Glu Thr Asn Ala Ala

210 215 220

Ala Ile Cys His Leu Tyr Gln Leu Ile Lys Gln Ala Met Met Thr Tyr

225 230 235 240

Gln Glu Gln Asn Arg Asn Leu Glu Leu Pro Asp Ser Gly Phe Val Ile

245 250 255

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

260 265 270

Ser Leu Glu Gly Phe Met Cys Ser Ser Phe Glu Phe Leu Ala Ala His

275 280 285

Leu Trp Lys Ala Arg Thr Arg Ala Leu Gly Leu Arg Arg Asp Ala Met

290 295 300

Val Cys Leu Gln Phe Ala Val Asp Ile Arg Lys Arg Thr Glu Thr Pro

305 310 315 320

Leu Pro Glu Gly Phe Ser Gly Asn Ala Tyr Val Leu Ala Ser Val Ala

325 330 335

Ser Thr Ala Arg Glu Leu Leu Glu Glu Leu Thr Leu Glu Ser Ile Val

340 345 350

Asn Lys Ile Arg Glu Ala Lys Lys Ser Ile Asp Gln Gly Tyr Ile Asn

355 360 365

Ser Tyr Met Glu Ala Leu Gly Gly Ser Asn Asp Gly Asn Leu Pro Pro

370 375 380

Leu Lys Glu Leu Thr Leu Ile Ser Asp Trp Thr Lys Met Pro Phe His

385 390 395 400

Asn Val Gly Phe Gly Asn Gly Gly Glu Pro Ala Asp Tyr Met Ala Pro

405 410 415

Leu Cys Pro Pro Val Pro Gln Val Ala Tyr Phe Met Lys Asn Pro Lys

420 425 430

Asp Ala Lys Gly Val Leu Val Arg Ile Gly Leu Asp Pro Arg Asp Val

435 440 445

Asn Gly Phe Ser Asn His Phe Leu Asp Cys

450 455

Claims (5)

1. the application of Arabidopis thaliana At4g31910 gene aspect improvement plant type of rice proterties, it is characterized in that adopting paddy rice Japan fine is transgene receptor, use the carrier pCAMBIA1306 with arabidopsis gene At4g31910, make up the mistake express transgenic plant of At4g31910 gene.

2. application according to claim 1, it is characterized in that the carrier pCAMBIA1306 with arabidopsis gene At4g31910, is that gene with Arabidopis thaliana At4g31910 is inserted between the KpnI in pCAMBIA1306 carrier 35S constitutive promoter downstream and the BamHI restriction enzyme site and obtains.

3. application according to claim 1, the clone who it is characterized in that arabidopsis gene At4g31910 obtains its amplification take the cDNA of Arabidopis thaliana as template by PCR.

4. application according to claim 1, the cDNA sequence that it is characterized in that arabidopsis gene At4g31910 is as described in the SEQ.ID.NO.1.

5. application according to claim 1, the protein sequence that it is characterized in that arabidopsis gene At4g31910 is as described in the SEQ.ID.NO.2.

Images