CN100422331C - A tufty serine/threonine kinase gene and its encoded protein - Google Patents

Abstract

The invention discloses a serine/threonine kinase gene (Hv-S/TPK) from Haynaldia villosa and its coded protein, belonging to the field of genetic engineering. The cDNA sequence of Serine/Threonine Kinase (Hv-S/TPK) of T. villosa is SEQ ID NO.1 and its coded amino acid sequence is SEQ ID NO.2. This gene is reported for the first time in O. villosa, and mRNA expression analysis shows that the expression of this gene is induced by powdery mildew; transgenic experiments show that the overexpression of this gene can improve the powdery mildew resistance of the powdery mildew variety Yangmai 158, and the transgenic plants are resistant to powdery mildew. Powdery mildew showed high resistance. Hv-S/TPK can be used as a target gene to introduce powdery mildew-susceptible wheat varieties to improve powdery mildew resistance.

Description

A tufty serine/threonine kinase gene and its encoded protein

1. Technical field:

The invention discloses a serine/threonine kinase gene (Hv-S/TPK) from wheat tufts, which belongs to the field of genetic engineering. The gene can be transferred into susceptible wheat to improve the powdery mildew resistance of wheat.

2. Technical background:

Wheat (Triticum asetivum L.) is the most widely distributed and planted food crop in the world. In my country, the planting area and output of wheat are next to rice, ranking second. Wheat has a long growth period and is susceptible to many biotic and abiotic stresses during its growth. Wheat powdery mildew caused by the obligate parasitic fungus Blumeria graminis f.sp.tritici is the second largest disease of wheat in my country. hazards. The most economical and effective way to control powdery mildew is to use disease-resistant varieties. However, due to the rapid change of pathogenic species, most of the existing varieties in my country have gradually lost their resistance to powdery mildew. The isolation and identification of new powdery mildew resistance genes, the use of genetic engineering to transfer into susceptible varieties, and the development of wheat disease-resistant molecular breeding can improve the disease resistance of wheat.

The Pm2l gene located on the 6V chromosome of T. villosa has strong resistance to powdery mildew and a broad spectrum of resistance, and is by far the most effective powdery mildew resistance gene (references: Chen P D, Qi L L, Zhou B, et al. Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew. Theor Appl Genet, 1995, 91: 1125-1128.), so cloning the gene and transferring it into susceptible varieties can improve wheat resistance. In recent years, our laboratory has carried out the work of cloning Pm2l, and identified the disease resistance function of the gene on the basis of screening the candidate genes.

3. Contents of the invention:

technical problem:

The purpose of the present invention is to disclose a serine/threonine kinase gene and its coded protein. The gene is from Trichophyllum villosa and can be introduced into common wheat as a target gene to improve wheat powdery mildew resistance and improve wheat varieties.

Technical solutions:

The serine/threonine kinase gene of the present invention is from Haynaldia villosa, named Hv-S/TPK gene, and its nucleotide sequence is SEQ ID NO.1. The protein encoded by the serine/threonine kinase gene is named Hv-S/TPK protein, and its amino acid sequence is SEQ ID NO.2.

Beneficial effect:

1. The present invention discloses a serine/threonine kinase gene (Hv-S/TPK) and its encoded protein. The serine/threonine kinase gene is the first report in G. villosa, which can be introduced into common wheat as a target gene to improve wheat powdery mildew resistance and improve wheat varieties.

2. Use the Hv-S/TPK gene of the present invention as the target gene to construct a plant expression vector, wherein any promoter such as cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promoter or other promoters can be used. To simplify the identification of transformed cells selectable markers can be used including enzymes that are resistant to antibiotics and compounds that can be identified by a color change (e.g. B-glucuronidase GUS) or luminescence (e.g. luciferase) Classes can also be selected with no markers. The transformation method uses the mature gene gun method of wheat transformation technology.

4. Description of drawings

Figure 1 Semi-quantitative RT-PCR of Hv-S/TPK gene

0, 24, 48, 72 means samples induced for 0, 24, 48, 72 hours

Figure 2 PCR with NAU/xibao15-F and NAU/xibao15-R as primers, and a set of DNA of Yangmai 5, Trichophyllum and Triticum villosa as templates

Note: Arrows indicate amplified bands from 6VS

Figure 3 PCR with NAU/xibao15-F and NAU/xibao15-R as primers, Yangmai 5, wheat-T. villosa DNA as template

Note: Arrows indicate amplified bands from 6VS

Figure 4 RACE amplification results

Note: The arrow shows the band of RACE

Fig. 5 Identification results of disease resistance of detached leaves

Fig. 6 Identification results of plant disease resistance under natural conditions

Figure 7: Chromosome diagram of common wheat

Figure 8: Schematic diagram of the chromosomes of T. villosa

Figure 9: Schematic representation of the wheat-P. villosa translocation line 6VS/6AL

Figure 10: Schematic representation of the wheat-T. villosa additive line DA6V

5. Specific implementation

1. Using barley gene chips to screen for disease-resistant gene analogs of T. villosa

The seeds of disease-resistant A. villosa (known public materials, Chen P D, Qi L L, Zhou B, et al. Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew. Theor Appl Genet, 1995, 91 : 1125-1128.) Sowed in a petri dish to germinate, transplanted to pots after dew white, and shake off the mixed powdery mildew spores (taken from the mixed spores of wheat powdery mildew in the field under natural conditions in Nanjing) on the seedlings at the three-leaf stage Induce on. The disease-resistant T. villosa was sampled 24, 48, and 72 hours after powdery mildew inoculation, and RNA was extracted separately (extracted with Trizol reagent from Invitrogen Company) and mixed in equal amounts to form an experimental group; non-induced leaves and induced samples were sampled at the same time, and RNA was extracted separately, etc. Amounts were mixed to form a control group. The RNA samples of the experimental group and the control group were reverse-transcribed into cDNA with Superscript ^TM II (the kit was purchased from Gibco/BRL, Gaithersburg, MD, USA), and further transcribed into cRNA in vitro. The cRNA samples of the experimental group and the control group were hybridized with Barley 1 GeneChip ( http://www.affymetrix.com/products/arrays/specific/barley.affx ) at the same time, and the chip hybridization experiment "Shanghai National Biochip Project Center" was completed, and the experimental steps were referred to "Expressed Analysis Technical Manual" of Affymetrix Company ( http://www.affymetrix.com/support/technical/manual/expression manual.affx ). The up-regulated expression genes were screened based on the ratio of the signal of the experimental group to the signal of the control group being greater than 2, and the disease resistance gene analog numbered Contig17515 was obtained, which is a serine/threonine kinase gene. Since the expression level of this gene is higher in the induced samples than in the non-induced samples, and the ratio of the hybridization signal of the experimental group to the hybridization signal of the control group is 2 ^1.7 , it is preliminarily judged that the gene is closely related to powdery mildew resistance.

2. Semi-quantitative RT-PCR analysis of gene expression

In order to verify the authenticity of the gene chip data, a pair of primers NAU/xibao15F (agatccaacaccagttcaag) and NAU/XiBao15R (atgttatggaggcttgtgtc) were designed according to the Contig17515 sequence for semi-quantitative RT-PCR analysis. The cDNA of non-induced and induced samples of disease-resistant T. villosa at different times were used as templates, NAU/xibao15 was used as primers for semi-quantitative RT-PCR analysis, and the housekeeping gene Tubulin was used as an internal reference (the primer for amplifying Tubulin gene was tubulinF- agaacactgttgtaaggctcaac and tubulinR-gagctttactgcctcgaacatgg). The PCR program was pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, renaturation at 56°C for 1min, extension at 72°C for 2min, 23 cycles; 10min at 72°C; storage at 4°C. The PCR product was electrophoresed on an agarose gel, and a band amplified by NAU/XiBao15 was detected in the T. villosa sample. After cloning and sequencing, the fragment was 448 bp long, and its homology with Contig17515 was 96%. Therefore, the cluster The gene fragment in hairy wheat was named Hv-S/TPK.

The results of semi-quantitative RT-PCR showed that: the expression of Hv-S/TPK gene was induced by powdery mildew in the disease-resistant T. villosa, and the expression result of RT-PCR (Figure 1) was consistent with the expression result of the gene chip. of.

3. Physical location of Hv-S/TPK gene

Using NAU/xibao15 as a primer, common wheat Yangmai No. 5 (a known and public approved variety), and wheat-Haynaldia (a known public, Chen P D, Qi L L, Zhou B, et al. Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance topowdery mildew. Theor Appl Genet, 1995, 91: 1125-1128.), a set of addition lines DA1V-DA7V (commonly known and public, see Sears, E.R.1953.Addition of the Genome of Haynaldiavillosa to Triticum aestivum.Am.J.Bot.40:168-174) DNA as a template for PCR, a specific band was amplified from Triticum villosa, which was only detected in DA6V in a set of addition lines This specific band of A. villosa (Fig. 2) indicates that the PCR product comes from the 6V chromosome of A. visicolor. After cloning and sequencing, the fragment is 902bp, and its left and right ends are consistent with the sequence of 448bp amplified from the cDNA of T. villosa. Further, common wheat Yangmai No. 5, 6VS/6AL translocation lines (known and public, Chen P D, Qi L L, Zhou B, et al. Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew.Theor Appl Genet, 1995, 91:1125-1128.) was used as a template for PCR, and a 902bp specific band was detected in the translocation line (Fig. 3). Because the 6AS chromosome in the 6VS/6AL translocation line was replaced by the 6VS chromosome of T. villosa, and a 902bp band could be amplified from the translocation line, the PCR product came from the 6VS chromosome. In summary, the Hv-S/TPK gene is located on the 6VS chromosome of T. villosa. (Note: The schematic diagrams of the chromosomes of the above materials are shown in Figures 7, 8, 9, and 10)

4. Using the RACE method to isolate the full length of the Hv-S/TPK gene

RACE was carried out using the cDNA of the disease-resistant T. villosa induced for 24 hours as a template, and the full-length cDNA of Hv-S/TPK was cloned using the SMART-RACE method (purchased from Clonetech, USA). 3'-RACE was isolated to 307bp at the 3' end of the gene, 5'-RACE was isolated to 621bp at the 5' end of the gene, and the full-length Hv-S/TPK gene of 1376bp was obtained after splicing (Figure 4). The primers Hv-S/TPK-F including the ORF frame were redesigned according to the spliced sequence: ATATAAGGGGCGGCTAAGGA and Hv-S/TPK-R: CACTGCGCCACCAATCTAGT. Using Hv-S/TPK-F and Hv-S/TPK-R as primers, PCR was carried out with the cDNA induced for 24 hours as a template, and a fragment of about 1200bp was detected (Figure 4), and the fragment was cloned and sequenced. Compared with the spliced sequence of 1376bp, the alignment result showed that the amplified sequence was consistent with the spliced sequence.

Using the full-length nucleotide sequence of the Hv-S/TPK gene to perform BLAST comparison analysis in the NCBI database, Hv-S/TPK is homologous to the serine/threonine kinase genes of barley, rice, Arabidopsis, and maize 96%, 87%, 81%, 79%, which further proves that Hv-S/TPK is a serine/threonine kinase gene.

5. Identification of gene function

Using Hv-S/TPK-F and Hv-S/TPK-R as primers to construct the gene fragment amplified from T. villosa into the transformation vector pAHC-25 (known and public, references: Christensen A H, Quail P H, Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable markergenes in monocotyledonous plants. Transgenic Research, 1996, 5: 213-218.), replace the GUS gene coding sequence on the pAHC25 vector with Hv-S/TPK , thus cloning the target gene to the downstream of the strong promoter Ubi to obtain the expression vector pAHC-Hv-S/TPK. The herbicide resistance gene (Bar gene) was used as a selectable marker gene for plant transformation.

Using young embryos and callus of Yangmai 158 (callus induction medium: MS+2, 4-D 2mg/L+ sucrose 30g/L+agar 8g/L, pH 5.8) as transformation recipients, the constructed expression The vector pAHC-Hv-S/TPK was introduced into the recipient by gene gun. The callus after bombardment is through two rounds of herbicide selection (selection medium is: MS+2,4-D 1mg/L+sucrose 30g/L+agar 8g/L+herbicide (Bialaphos) 3-5mg/L, pH 5.8 ), the resistant callus was regenerated into resistant plants on the differentiation medium (the differentiation medium was: 1/2MS+naphthalene acetic acid 1.0mg/L+herbicide (Bialaphos) 34mg/L+30g/L sucrose+agar 8g, PH5 .8). The total DNA of leaves of regenerated plants was extracted for PCR detection. If the three genes of herbicide resistance gene (Bar), promoter gene (Ubi) and target gene (Hv-S/TPK) were all detected, the regenerated plants were regarded as positive plants.

The disease resistance function of the positive plants was identified. (1) Identification of disease resistance of detached leaves of regenerated seedlings. Put the detached leaves on 2.4% agar powder plus 20mg/L benzimidazole fresh-keeping medium, inoculate fresh powdery mildew spores, culture in the dark at 18-20°C within 24 hours after inoculation, and then culture in weak light for 7 days Investigate the outcome of the disease. The identification results showed that there were only allergic spots on the transgenic plants and no visible spores; while the powdery mildew spores were all over the whole leaves on the control leaves (Fig. 5). (2) After the seedlings survived the summer in the artificial climate chamber, they were transferred to the field, and the disease resistance of the transgenic plants was identified under natural disease conditions. The results of the field identification were consistent with those of the detached leaves. There were no spore piles on the transgenic plants, but there were spore piles on the leaves of the control plants ( FIG. 6 ). Combining the results of detached leaves and field identification, the transgenic plants were highly resistant to powdery mildew, indicating that Hv-S/TPK has disease resistance function.

The sequences and symbols involved in the present invention are listed as follows:

(1) Information on SEQ ID NO.1

(i) Sequential features:

(A) Length: 1376bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: Linear

(ii) Molecule type: Nucleotide

(iii) Sequence description: SEQ ID NO.1

(2) Information of SEQ ID NO.2

(i) Sequential features:

(A) Length: 310a.a

(B) Type: amino acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecule type: protein

(iii) Sequence description: SEQ ID NO.2

sequence listing

<110> Nanjing Agricultural University

<120>A Serine/Threonine Kinase Gene and Its Encoded Protein

<130>000

<140>000

<141>2006-06-21

<160>3

<170>PatentIn version 3.1

<210>SEQ ID NO.1

<211>1376

<212>DNA

<213> tuft wheat [Haynaldia villosa]

<221> CDS

<222>(163)..(1095)

<221>3′UTR

<222>(1096)..(1376)

<221>mRNA

<222>(1)..(1376)

<221>polyA_site

<222>(1360)..(1376)

<221>5′UTR

<222>(1)..(162)

<400>1

gagctagcaa aggcaacaga aaattttaac ccctccaaca agattggtga gggggtttt 60

ggatctgtat ataaggggcg gctaaggaat ggaaaactta ttgctgtcaa ggtgttatct 120

gtagaatcaa gacaaggatt aaaggagttt ctgaatgaac tgatgtcaat ttccaacata 180

tctcatggca atcttgtcag cctttatggc tattgtgtgg aaggaaacca gaggatcctt 240

gtttacaatt accttgagaa taatagccta gcacaaacac ttctaggttc tggccgcagc 300

aatatccagt tcaattggag aagtagagta aatatttgcc ttggtatcgc ccgaggatta 360

gcataccttc atgatgatgt caatccccac attgttcatc gggatatcaa agcaagcaat 420

atacttcttg ataaggatct cacccccaaa atttctgatt tcggtttagc aaagcttcta 480

cctccaaatg cgtcacatat tagcacacgg gttgcaggaa cattaggcta cttggctcct 540

gagtatgcca ttcgaggaca agtgacacgg aagtcagatg tttatagttt tggtgttttg 600

cttctggaaa tagtcagtgg gagatccaac accagttcaa gattacccta tgaagaccaa 660

atacttttgg aaaagttccc agaggttacc aatggggttc tcctcttgca gacatggatg 720

tattatgagc agggagattt ggtgaaaatc atagacagtt ctgtgggtga tgatttggat 780

attgaacaag cctgcaggtt cctgaaagtt ggacttctct gtacacaaga tgtcacaaga 840

catcgaccca ccatgtcaac tgtcgtcagc atgctagcag gcgaaaagga tgttgactcg 900

gagaagatca gcaagcccgc tacaattagc gactttatgg acctcaagat caggagcatg 960

aggagagaaa ataacatagc tttcgcttct tcctccacgt tgctatccac tatcatggca 1020

cactcttctc cattgttgtc gcaagagacg acacaagcct ccataaatt caccgcaata 1080

tcagagcgtg agtgacctga agttggttgc agatacgaag accatgtaga gaagtagcat 1140

agccagatac cttttctttt tcagaaaatt tcttcctagt gtatagattc actttgttta 1200

tagtgtacac agcattgctg gcacaaagaa gttaaccagt tttactttgt gtgtataata 1260

ctagattggt ggcgcagtgt cattgtataa tttgtgcatg tacatcgtgt ctgttgtttg 1320

tttggagggt aagaaaatac aagtttgaga tcctgtgagt aaaaaaaaaa aaaaaa 1376

<210>SEQ ID NO.2

<211>310

<212>PRT

<213> Haynaldia villosa

<400>2

Met Ser Ile Ser Asn Ile Ser His Gly Asn Leu Val Ser Leu Tyr Gly

1 5 10 15

Tyr Cys Val Glu Gly Asn Gln Arg Ile Leu Val Tyr Asn Tyr Leu Glu

20 25 30

Asn Asn Ser Leu Ala Gln Thr Leu Leu Gly Ser Gly Arg Ser Asn Ile

35 40 45

Gln Phe Asn Trp Arg Ser Arg Val Asn Ile Cys Leu Gly Ile Ala Arg

50 55 60

Gly Leu Ala Tyr Leu His Asp Asp Val Asn Pro His Ile Val His Arg

65 70 75 80

Asp Ile Lys Ala Ser Asn Ile Leu Leu Asp Lys Asp Leu Thr Pro Lys

85 90 95

Ile Ser Asp Phe Gly Leu Ala Lys Leu Leu Pro Pro Asn Ala Ser His

100 105 110

Ile Ser Thr Arg Val Ala Gly Thr Leu Gly Tyr Leu Ala Pro Glu Tyr

115 120 125

Ala Ile Arg Gly Gln Val Thr Arg Lys Ser Asp Val Tyr Ser Phe Gly

130 135 140

Val Leu Leu Leu Glu Ile Val Ser Gly Arg Ser Asn Thr Ser Ser Arg

145 150 155 160

Leu Pro Tyr Glu Asp Gln Ile Leu Leu Glu Lys Phe Pro Glu Val Thr

165 170 175

Asn Gly Val Leu Leu Leu Gln Thr Trp Met Tyr Tyr Glu Gln Gly Asp

180 185 190

Leu Val Lys Ile Ile Asp Ser Ser Val Gly Asp Asp Leu Asp Ile Glu

195 200 205

Gln Ala Cys Arg Phe Leu Lys Val Gly Leu Leu Cys Thr Gln Asp Val

210 215 220

Thr Arg His Arg Pro Thr Met Ser Thr Val Val Ser Met Leu Ala Gly

225 230 235 240

Glu Lys Asp Val Asp Ser Glu Lys Ile Ser Lys Pro Ala Thr Ile Ser

245 250 255

Asp Phe Met Asp Leu Lys Ile Arg Ser Met Arg Arg Glu Asn Asn Ile

260 265 270

Ala Phe Ala Ser Ser Ser Thr Leu Leu Ser Thr Ile Met Ala His Ser

275 280 285

Ser Pro Leu Leu Ser Gln Glu Thr Thr Gln Ala Ser Ile Thr Phe Thr

290 295 300

Ala Ile Ser Glu Arg Glu

305 310

<210>3

<211>310

<212>PRT

<213> tuft wheat [Haynaldia villosa]

<221>Hv-S/TPK

<222>(1)..(310)

<400>3

Met Ser Ile Ser Asn Ile Ser His Gly Asn Leu Val Ser Leu Tyr Gly

1 5 10 15

Tyr Cys Val Glu Gly Asn Gln Arg Ile Leu Val Tyr Asn Tyr Leu Glu

20 25 30

Asn Asn Ser Leu Ala Gln Thr Leu Leu Gly Ser Gly Arg Ser Asn Ile

35 40 45

Gln Phe Asn Trp Arg Ser Arg Val Asn Ile Cys Leu Gly Ile Ala Arg

50 55 60

Gly Leu Ala Tyr Leu His Asp Asp Val Asn Pro His Ile Val His Arg

65 70 75 80

Asp Ile Lys Ala Ser Asn Ile Leu Leu Asp Lys Asp Leu Thr Pro Lys

85 90 95

Ile Ser Asp Phe Gly Leu Ala Lys Leu Leu Pro Pro Asn Ala Ser His

100 105 110

Ile Ser Thr Arg Val Ala Gly Thr Leu Gly Tyr Leu Ala Pro Glu Tyr

115 120 125

Ala Ile Arg Gly Gln Val Thr Arg Lys Ser Asp Val Tyr Ser Phe Gly

130 135 140

Val Leu Leu Leu Glu Ile Val Ser Gly Arg Ser Asn Thr Ser Ser Arg

145 150 155 160

Leu Pro Tyr Glu Asp Gln Ile Leu Leu Glu Lys Phe Pro Glu Val Thr

165 170 175

Asn Gly Val Leu Leu Leu Gln Thr Trp Met Tyr Tyr Glu Gln Gly Asp

180 185 190

Leu Val Lys Ile Ile Asp Ser Ser Val Gly Asp Asp Leu Asp Ile Glu

195 200 205

Gln Ala Cys Arg Phe Leu Lys Val Gly Leu Leu Cys Thr Gln Asp Val

210 215 220

Thr Arg His Arg Pro Thr Met Ser Thr Val Val Ser Met Leu Ala Gly

225 230 235 240

Glu Lys Asp Val Asp Ser Glu Lys Ile Ser Lys Pro Ala Thr Ile Ser

245 250 255

Asp Phe Met Asp Leu Lys Ile Arg Ser Met Arg Arg Glu Asn Asn Ile

260 265 270

Ala Phe Ala Ser Ser Ser Thr Leu Leu Ser Thr Ile Met Ala His Ser

275 280 285

Ser Pro Leu Leu Ser Gln Glu Thr Thr Gln Ala Ser Ile Thr Phe Thr

290 295 300

Ala Ile Ser Glu Arg Glu

305 310

Claims (2)

1. A serine/threonine kinase gene from Haynaldia villosa named Hv-S/TPK gene, its nucleotide sequence is SEQ ID NO.1. 2. The protein encoded by the serine/threonine kinase gene as claimed in claim 1 is called Hv-S/TPK protein, and its amino acid sequence is SEQ ID NO.2.

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