簇毛麦病毒诱导基因沉默体系的建立及其在基因功能研究中的应用
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摘要
二倍体簇毛麦(Haynaldia villosa Schur.,syn.Dasypyrum Villosum Candargy, 2n=2X=14.VV)是小麦抗病育种的重要基因源。簇毛麦6V染色体短臂上携带有高抗白粉病的主效基因Pm21,该基因抗谱广,抗性强。
为了研究Pmm21基因对白粉病的抗性机理并筛选簇毛麦抗白粉病相关基因,Cao等利用接种白粉菌后的抗、感簇毛麦和未接种的抗病簇毛麦为材料,将其cRNA与大麦基因芯片杂交,筛选出抗病簇毛麦经白粉菌诱导表达的基因,以及白粉菌诱导后抗、感簇毛麦的差异表达基因。本研究根据基因芯片上调表达的10条探针设计引物,在抗病簇毛麦cDNA中扩增,获得6个抗病相关基因的cDNA片段。其中,根据探针Contig3687设计引物,扩增获得的cDNA片段与水稻(Oryza sativa, Japonica Group)乙烯反应元件结合蛋白转录因子序列具有84%的一致性；根据探针Contig5017设计引物,扩增获得的cDNA片段与水稻(Oryza sativa,Japonica cultivar group)蛋白激酶互作子1基因具有90%一致性。利用cDNA片段扩增引物通过PCR反应筛选白粉菌诱导后抗病簇毛麦的cDNA文库,获得了包含全长乙烯反应元件结合蛋白(Ethylene responsive element binding protein,Hv-EREBP)(GenBank accession FJ711058)基因及蛋白激酶互作子1(Protein kinase interactor,Hv-PKI)(GenBank accession FJ711059)基因的cDNA文库单克隆。
测序结果表明,包含Hv-EREBP全长基因的cDNA文库单克隆插入片段长1429bp,其中包含一个完整的Hv-EREBP基因的开放阅读框,长1185bp。其推导的蛋白质序列编码394个氨基酸,具有一个完整的AP2保守结构域,与水稻转录因子EREBP (GenBank accession BAD19536)具有66%一致性。构建Hv-EREBP全长基因、该基因起始密码子与AP2保守结构域之间的序列分别与GFP基因重组的质粒,利用基因枪瞬间表达融合蛋白,检测融合蛋白在洋葱表皮细胞中的亚细胞定位。结果表明,EREBP蛋白为核蛋白,该蛋白在洋葱表皮细胞内的准确定位主要由起始密码子与AP2保守结构域之间的氨基酸序列决定利用pET原核表达系统,构建全长Hv-EREBP基因与pET32a载体重组的质粒,将该质粒转化大肠杆菌BL21(DE3)感受态细胞中。利用IPTG诱导Hv-EREBP基因与His标签重组的融合蛋白表达。结果表明,与对照相比,IPTG浓度依次为0.2mM、0.6mM、1.0mM、1.5mM和2.0mM时,转化了重组质粒的大肠杆菌均表达出一条特异的蛋白条带,分子量约为66.8KD,与Hv-EREBP融合蛋白的理论分子量相符。实时荧光定量RT-PCR分析表明,Hv-EREBP基因在白粉菌诱导下强烈上调表达。外源激素乙烯可以显著诱导Hv-EREBP基因的表达,水杨酸可以抑制Hv-EREBP基因的表达,而外源茉莉酸处理后该激素的表达量逐渐下降。推测Hv-EREBP基因可能与簇毛麦抗白粉病相关途径及乙烯防卫反应途径有关。
测序结果表明,包含Hv-PKI全长基因的cDNA文库单克隆插入片段长1519bp,其中包含一个完整的Hv-PKI基因的开放阅读框,长1089bp。其推导的蛋白质序列编码362个氨基酸,具有一个完整的蛋白激酶结构域,与水稻蛋白激酶互作子基因(GenBank accession AAS98413)具有93%一致性。构建Hv-PKI全长基因、该基因起始密码子与保守结构域之间的序列分别与GFP基因重组的质粒,利用基因枪瞬间表达融合蛋白,检测融合蛋白在洋葱表皮细胞中的亚细胞定位。结果表明,PKI蛋白为细胞质内和细胞膜上表达的蛋白,该蛋白在洋葱表皮细胞内的准确定位主要由起始密码子与保守结构域之间的氨基酸序列决定。利用pET原核表达系统,构建全长Hv-PKI基因与pET32a载体重组的质粒,将该质粒转化大肠杆菌BL21(DE3)感受态细胞中。利用IPTG诱导Hv-PKI基因与His标签重组的融合蛋白表达。结果表明,与对照相比,IPTG浓度依次为0.2mM、0.6mM、1.0mM、1.5mM和2.0mM时,转化了重组质粒的大肠杆菌均表达出一条特异的蛋白条带,分子量约为63.5KD,与Hv-PKI融合蛋白的理论分子量相符。实时荧光定量RT-PCR分析表明,白粉菌诱导及施加外源激素水杨酸可以显著诱导Hv-PK1基因在簇毛麦叶片中的表达,乙烯与茉莉酸对该基因的表达没有明显的作用。推测该基因可能与簇毛麦抗白粉病反应途径及水杨酸防卫反应途径有关。
Chen等以携带有Pm21的小麦近等基因系及其轮回亲本扬麦5号为材料,利用抑制性消减杂交技术,得到一个在抗病材料中特异表达的抗病基因类似物Ta-LRR2 (GeneBank accession ABQ53156.1),受白粉菌诱导后上调表达。染色体定位分析表明它位于麦类作物第六部分同源群染色体短臂的中部,根据该基因开发的分子标记CINAU16(NAU/XiBao16)与Pm21基因共分离(Chen等,2006)。本研究采用同源克隆方法,在簇毛麦中获得了一个编码226个氨基酸的抗病基因类似物Hv-LRR (GenBank accession FJ711057),定位在簇毛麦6VS染色体上。实时荧光定量RT-PCR分析表明,白粉菌诱导后Hv-LRR强烈上调表达,推测该基因可能在簇毛麦叶片中参与抗白粉病相关反应途径。
簇毛麦转化体系尚不成熟。为了促进簇毛麦基因功能研究,利用大麦条花叶病毒(Barley stripe mosaic virus,BSMV)载体在簇毛麦中成功构建了稳定有效的基因沉默体系。接种BSMV:GFP重组病毒后,绿色荧光蛋白(Green fluorescent protein,GFP)基因表达观察的结果表明,BSMV传递过程在簇毛麦生长早期即可发生；接种BSMV:PDS重组病毒,沉默簇毛麦八氢番茄红素脱饱和酶(Phytoene desaturase,PDS)基因后,簇毛麦植株表型观察与Real-time RT-PCR检测结果表明,携带PDS基因片段的BSMV重组病毒可以在簇毛麦中进行系统侵染,6天后即可引起PDS基因表达明显下调。PDS基因沉默造成的叶片白化表型主要发生在植株上部新生叶片,沉默效果可以稳定维持于整个生育期。该体系的构建,为利用病毒诱导基因沉默(Virus induced gene silencing,VIGS)技术进行簇毛麦基因功能研究提供了工具。
利用所构建的基因沉默体系对本研究克隆的Hv-EREBP基因及本实验室克隆的丝氨酸/苏氨酸蛋白激酶(Serine/threonine protein kinase,Hv-S/TPK)基因进行了功能研究,Real-time RT-PCR检测结果表明,携带目标基因片段的BSMV重组病毒可以有效沉默相应的基因,发生了基因沉默的簇毛麦叶片上白粉菌侵染率增加,簇毛麦抗性有所减弱。表明这两个基因在簇毛麦抗白粉病相关途径中发挥了作用。
Haynaldia villosa Schur (2n=14, VV), whose synonymous name is Dasypyrum villosum Candargy, is a diploid relative of wheat. It is an important genetic resource for wheat disease resistance breeding. Especially, on its short arm of 6V chromosome, a major gene Pm21 with high powdery mildew resistance has been transferred to wheat in breeding programs successfully.
To clone genes involved in resistance to powdery mildew and investigate resistance mechanism in H.villosa, cDNA from the resistant H.villosa and susceptible mutant both inoculated with Blumeria gramimis f.sp. tritici Marchal (ab. Bgf), a fungus who can cause powdery mildew, and cDNA from the uninoculated resistant H. villosa were hybridized to the Barley 1 microarrays respectively in our laboratory. And those genes that were significantly up-regulated expressed in inoculated resistant H.villosa or differently expressed between inoculated resistant and susceptible plant were identified. Based on information of these differential expressed genes, ten disease relative gene sequences corresponding to ten up-regulated probes were selected as templates for PCR primer pairs designation, and PCR amplification were performed in cDNA of H. villosa inoculated with Bgt. Six cDNA fragments were obtained out of ten. Among these cDNA fragments, the one amplified with primers designated from probe Contig3687 showed 84% identity with O.sativa (Japonica Group) EREBP transcription factor, and the one amplified with primers designated for Contig 5017 showed 90% identity with O.sativa(japonica cultivar group) pto kinase interactor 1. Then the two primer pairs were screened independently in cDNA library made from resistant H.villosa inoculated with Bgt, and clones containing inserts representing a full length Hv-EREBP (Ethylene responsive element binding protein, EREBP) (GenBank accession FJ711058) gene and a full length Hv-PKI (Protein kinase interactor, PKI) (GenBank accession FJ711059) gene were obtained respectively.
Tne clone containing a full length Hv-EREBP gene has a 1429bp insert, in which an Hv-EREBP gene with a 1185bp complete ORF was identified. Composed of 394 amino acids, the deduced protein of this Hv-EREBP gene encoded a complete AP2 domain. This protein has a 66% identity with O.sativa transcription factor EREBP1 (GenBank accession BAD 19536). Subcellular localization analysis showed that EREBP-GFP fusion proteins expressed intensively in the cell nucleus of onin epidermal cells, and the CDS fragment from the start code to the AP2 coserved domain encoded the amino adid sequence required for accurate positioning of this protein. Expression of the fusion protein Hv-EREBP was carried out with the pET expression system successfully, a powerful system developed for the cloning and expression of recombinant proteins in protease deficienct bacterial strains, i.e. E. coli. BL21(DE3). Recombinant plasmid was constructed by insertion of the full length Hv-EREBP gene into the pET32a vector, and transformed into the competent host cell BL21(DE3). After induction with the addition of IPTG to the bacterial culture, compared with the controls, the host stains contained the recombinant plasmid has yielded a specific band whose molecular weight was equal to the deduced theoretical molecular weight of the fusion protein Hv-EREBP, i.e. about 66.8KD. Real-time RT-PCR analysis showed that transcription of the Hv-EREBP gene was up-regulated significantly in H.villosa after inoculation with Bgt or application with exogenous ethylene, the gene transcription was suppressed in H.villosa treated with exogenous salicylic acid, and transcripts of this gene decreased gradually in H. villosa sprayed with exogenous jasmonic acid. Deduced from above observation, the Hv-EREBP gene must play a role in powdery mildew resistant pathway or ethylene defense pathway in H.villosa.
Tne clone containing a full length Hv-PKI gene has a 1519bp insert, in which a 1089bp complete ORF representing a full length Hv-PKI gene was identified. The Hv-PKI gene encoded a deduced protein composed with 362 amino acids, in which a complete protein kinase domain was identified. This deduced protein has 93% identity with O.sativa protein kinase interactor (GenBank accession AAS98413). Subcellular localization analysis showed that PKI-GFP fusion proteins expressed intensively in the cytoplasm and on the membrane of onin epidermal cells, the CDS fragment from the start code to the coserved domain encoded the amino adid sequence required for accurate positioning of this protein. Expression of the fusion protein Hv-PKI was carried out with the pET expression system successfully. Recombinant plasmid was constructed by insertion of the full length Hv-PKI gene into the pET32a vector, and transformed into the competent host cell BL21(DE3). After induction by IPTG, compared with the controls, the host stains carrying the recombinant plasmid has produced a unique band whose molecular weight was equal to the deduced molecular weight of Hv-PKI protein, i.e.63.3KD. Analyzed by Real-time RT-PCR, expression of this gene was induced remarkably by exogenous hormone salicylic acid or Bgt. On the contrary, no sharp expression increase or decrease was detected in leaves of H.villosa after application with exogenous ethylene or jasmonic acid. Deduced from above observation, this gene must involve in powdery mildew reisitance and salicylic acid defense pathway in H.villosa.
To clone candidate gene for Pm21, suppression subtractive hybridization was conducted between an isogenic resistant line carrying Pm21 and its recurrent parent Yangmai 5 by Chen et al., and a resistance gene analog (RGA) Ta-LRR2 (GeneBank accession ABQ53156.1) co-segregating with Pm21 was obtained. Afterwards, a resistance gene analog polymorphic molecular marker CINAU16 (NAU/XiBao 16) linked to Pm21 was developed from Ta-LRR2 sequence. Ta-LRR2 had an increased expression level in the resistant wheat T6VS/6AL after inoculation with Bgt. In this work, a homologous RGA sequence Hv-LRR encoding 226 amino acids (GenBank accession FJ711057) was cloned, assigned on the short arm of 6V chromosome in H. villosa. Expression of the Hv-LRR was dramatically induced in leaves of H.villosa challenged by Bgt, but not induced by exogenous hormones, i.e. ethylene, salicylic acid, jasmonic acid. This gene was deduced taking part in the powdery mildew resistant pathway in H. villosa.
From our experience, tissue culture system for H.villosa was still quite difficult to be established at present time. To fast evaluate gene function, an effective and persistent virus-induced gene silencing (VIGS) system was established with barley stripe mosaic virus (BSMV) for H.villosa. Fluorescence observation of GFP gene expression showed that BSMV:GFP can deliver systemically from leaf to leaf in the early growing stage of the plant after inoculation. Characterization of phytoene desaturase (PDS) gene silencing process and Real-time RT-PCR detection showed that BSMV:PDS carrying reverse inserted fragment of PDS gene can infect H.villosa systemically, and suppress PDS transcripts as early as six days after inoculation. The photobleaching phenotype occurred mainly on the newly upper leaves, and PDS gene silencing process carried thorough the whole growing period in H.villosa. This was the first report that BSMV was used successfully for VIGS in a wild relative species of wheat. The established VIGS system will surely be a powerful reverse genetics tool for gene function study in H.villosa.
With the established VIGS system, function analysis of two genes, i.e. the Hv-EREBP gene and a serine/threonine protein kinase(Hv-S/TPK) gene, were carried out. Cloned by Cao et al. in our laboratory previously, the Hv-S/TPK gene was a candidate gene for Pm21, and located on the short arm of 6 V chromosome in H. villosa. Expression of the Hv-EREBP gene or the Hv-S/TPK gene was dramatically down-regulated in plants inoculated with BSMV:Hv-EREBP or BSMV:Hv-S/TPK. Infection percentages by Bgt increased on leaves of H. villosa in which Hv-EREBP or Hv-S/TPK were silenced, and resistance to powdery mildew decreased. All these results showed that Hv-EREBP and Hv-S/TPK definitely played a role in powdery mildew resistant pathway in H.villosa.
为了研究Pmm21基因对白粉病的抗性机理并筛选簇毛麦抗白粉病相关基因,Cao等利用接种白粉菌后的抗、感簇毛麦和未接种的抗病簇毛麦为材料,将其cRNA与大麦基因芯片杂交,筛选出抗病簇毛麦经白粉菌诱导表达的基因,以及白粉菌诱导后抗、感簇毛麦的差异表达基因。本研究根据基因芯片上调表达的10条探针设计引物,在抗病簇毛麦cDNA中扩增,获得6个抗病相关基因的cDNA片段。其中,根据探针Contig3687设计引物,扩增获得的cDNA片段与水稻(Oryza sativa, Japonica Group)乙烯反应元件结合蛋白转录因子序列具有84%的一致性；根据探针Contig5017设计引物,扩增获得的cDNA片段与水稻(Oryza sativa,Japonica cultivar group)蛋白激酶互作子1基因具有90%一致性。利用cDNA片段扩增引物通过PCR反应筛选白粉菌诱导后抗病簇毛麦的cDNA文库,获得了包含全长乙烯反应元件结合蛋白(Ethylene responsive element binding protein,Hv-EREBP)(GenBank accession FJ711058)基因及蛋白激酶互作子1(Protein kinase interactor,Hv-PKI)(GenBank accession FJ711059)基因的cDNA文库单克隆。
测序结果表明,包含Hv-EREBP全长基因的cDNA文库单克隆插入片段长1429bp,其中包含一个完整的Hv-EREBP基因的开放阅读框,长1185bp。其推导的蛋白质序列编码394个氨基酸,具有一个完整的AP2保守结构域,与水稻转录因子EREBP (GenBank accession BAD19536)具有66%一致性。构建Hv-EREBP全长基因、该基因起始密码子与AP2保守结构域之间的序列分别与GFP基因重组的质粒,利用基因枪瞬间表达融合蛋白,检测融合蛋白在洋葱表皮细胞中的亚细胞定位。结果表明,EREBP蛋白为核蛋白,该蛋白在洋葱表皮细胞内的准确定位主要由起始密码子与AP2保守结构域之间的氨基酸序列决定利用pET原核表达系统,构建全长Hv-EREBP基因与pET32a载体重组的质粒,将该质粒转化大肠杆菌BL21(DE3)感受态细胞中。利用IPTG诱导Hv-EREBP基因与His标签重组的融合蛋白表达。结果表明,与对照相比,IPTG浓度依次为0.2mM、0.6mM、1.0mM、1.5mM和2.0mM时,转化了重组质粒的大肠杆菌均表达出一条特异的蛋白条带,分子量约为66.8KD,与Hv-EREBP融合蛋白的理论分子量相符。实时荧光定量RT-PCR分析表明,Hv-EREBP基因在白粉菌诱导下强烈上调表达。外源激素乙烯可以显著诱导Hv-EREBP基因的表达,水杨酸可以抑制Hv-EREBP基因的表达,而外源茉莉酸处理后该激素的表达量逐渐下降。推测Hv-EREBP基因可能与簇毛麦抗白粉病相关途径及乙烯防卫反应途径有关。
测序结果表明,包含Hv-PKI全长基因的cDNA文库单克隆插入片段长1519bp,其中包含一个完整的Hv-PKI基因的开放阅读框,长1089bp。其推导的蛋白质序列编码362个氨基酸,具有一个完整的蛋白激酶结构域,与水稻蛋白激酶互作子基因(GenBank accession AAS98413)具有93%一致性。构建Hv-PKI全长基因、该基因起始密码子与保守结构域之间的序列分别与GFP基因重组的质粒,利用基因枪瞬间表达融合蛋白,检测融合蛋白在洋葱表皮细胞中的亚细胞定位。结果表明,PKI蛋白为细胞质内和细胞膜上表达的蛋白,该蛋白在洋葱表皮细胞内的准确定位主要由起始密码子与保守结构域之间的氨基酸序列决定。利用pET原核表达系统,构建全长Hv-PKI基因与pET32a载体重组的质粒,将该质粒转化大肠杆菌BL21(DE3)感受态细胞中。利用IPTG诱导Hv-PKI基因与His标签重组的融合蛋白表达。结果表明,与对照相比,IPTG浓度依次为0.2mM、0.6mM、1.0mM、1.5mM和2.0mM时,转化了重组质粒的大肠杆菌均表达出一条特异的蛋白条带,分子量约为63.5KD,与Hv-PKI融合蛋白的理论分子量相符。实时荧光定量RT-PCR分析表明,白粉菌诱导及施加外源激素水杨酸可以显著诱导Hv-PK1基因在簇毛麦叶片中的表达,乙烯与茉莉酸对该基因的表达没有明显的作用。推测该基因可能与簇毛麦抗白粉病反应途径及水杨酸防卫反应途径有关。
Chen等以携带有Pm21的小麦近等基因系及其轮回亲本扬麦5号为材料,利用抑制性消减杂交技术,得到一个在抗病材料中特异表达的抗病基因类似物Ta-LRR2 (GeneBank accession ABQ53156.1),受白粉菌诱导后上调表达。染色体定位分析表明它位于麦类作物第六部分同源群染色体短臂的中部,根据该基因开发的分子标记CINAU16(NAU/XiBao16)与Pm21基因共分离(Chen等,2006)。本研究采用同源克隆方法,在簇毛麦中获得了一个编码226个氨基酸的抗病基因类似物Hv-LRR (GenBank accession FJ711057),定位在簇毛麦6VS染色体上。实时荧光定量RT-PCR分析表明,白粉菌诱导后Hv-LRR强烈上调表达,推测该基因可能在簇毛麦叶片中参与抗白粉病相关反应途径。
簇毛麦转化体系尚不成熟。为了促进簇毛麦基因功能研究,利用大麦条花叶病毒(Barley stripe mosaic virus,BSMV)载体在簇毛麦中成功构建了稳定有效的基因沉默体系。接种BSMV:GFP重组病毒后,绿色荧光蛋白(Green fluorescent protein,GFP)基因表达观察的结果表明,BSMV传递过程在簇毛麦生长早期即可发生；接种BSMV:PDS重组病毒,沉默簇毛麦八氢番茄红素脱饱和酶(Phytoene desaturase,PDS)基因后,簇毛麦植株表型观察与Real-time RT-PCR检测结果表明,携带PDS基因片段的BSMV重组病毒可以在簇毛麦中进行系统侵染,6天后即可引起PDS基因表达明显下调。PDS基因沉默造成的叶片白化表型主要发生在植株上部新生叶片,沉默效果可以稳定维持于整个生育期。该体系的构建,为利用病毒诱导基因沉默(Virus induced gene silencing,VIGS)技术进行簇毛麦基因功能研究提供了工具。
利用所构建的基因沉默体系对本研究克隆的Hv-EREBP基因及本实验室克隆的丝氨酸/苏氨酸蛋白激酶(Serine/threonine protein kinase,Hv-S/TPK)基因进行了功能研究,Real-time RT-PCR检测结果表明,携带目标基因片段的BSMV重组病毒可以有效沉默相应的基因,发生了基因沉默的簇毛麦叶片上白粉菌侵染率增加,簇毛麦抗性有所减弱。表明这两个基因在簇毛麦抗白粉病相关途径中发挥了作用。
Haynaldia villosa Schur (2n=14, VV), whose synonymous name is Dasypyrum villosum Candargy, is a diploid relative of wheat. It is an important genetic resource for wheat disease resistance breeding. Especially, on its short arm of 6V chromosome, a major gene Pm21 with high powdery mildew resistance has been transferred to wheat in breeding programs successfully.
To clone genes involved in resistance to powdery mildew and investigate resistance mechanism in H.villosa, cDNA from the resistant H.villosa and susceptible mutant both inoculated with Blumeria gramimis f.sp. tritici Marchal (ab. Bgf), a fungus who can cause powdery mildew, and cDNA from the uninoculated resistant H. villosa were hybridized to the Barley 1 microarrays respectively in our laboratory. And those genes that were significantly up-regulated expressed in inoculated resistant H.villosa or differently expressed between inoculated resistant and susceptible plant were identified. Based on information of these differential expressed genes, ten disease relative gene sequences corresponding to ten up-regulated probes were selected as templates for PCR primer pairs designation, and PCR amplification were performed in cDNA of H. villosa inoculated with Bgt. Six cDNA fragments were obtained out of ten. Among these cDNA fragments, the one amplified with primers designated from probe Contig3687 showed 84% identity with O.sativa (Japonica Group) EREBP transcription factor, and the one amplified with primers designated for Contig 5017 showed 90% identity with O.sativa(japonica cultivar group) pto kinase interactor 1. Then the two primer pairs were screened independently in cDNA library made from resistant H.villosa inoculated with Bgt, and clones containing inserts representing a full length Hv-EREBP (Ethylene responsive element binding protein, EREBP) (GenBank accession FJ711058) gene and a full length Hv-PKI (Protein kinase interactor, PKI) (GenBank accession FJ711059) gene were obtained respectively.
Tne clone containing a full length Hv-EREBP gene has a 1429bp insert, in which an Hv-EREBP gene with a 1185bp complete ORF was identified. Composed of 394 amino acids, the deduced protein of this Hv-EREBP gene encoded a complete AP2 domain. This protein has a 66% identity with O.sativa transcription factor EREBP1 (GenBank accession BAD 19536). Subcellular localization analysis showed that EREBP-GFP fusion proteins expressed intensively in the cell nucleus of onin epidermal cells, and the CDS fragment from the start code to the AP2 coserved domain encoded the amino adid sequence required for accurate positioning of this protein. Expression of the fusion protein Hv-EREBP was carried out with the pET expression system successfully, a powerful system developed for the cloning and expression of recombinant proteins in protease deficienct bacterial strains, i.e. E. coli. BL21(DE3). Recombinant plasmid was constructed by insertion of the full length Hv-EREBP gene into the pET32a vector, and transformed into the competent host cell BL21(DE3). After induction with the addition of IPTG to the bacterial culture, compared with the controls, the host stains contained the recombinant plasmid has yielded a specific band whose molecular weight was equal to the deduced theoretical molecular weight of the fusion protein Hv-EREBP, i.e. about 66.8KD. Real-time RT-PCR analysis showed that transcription of the Hv-EREBP gene was up-regulated significantly in H.villosa after inoculation with Bgt or application with exogenous ethylene, the gene transcription was suppressed in H.villosa treated with exogenous salicylic acid, and transcripts of this gene decreased gradually in H. villosa sprayed with exogenous jasmonic acid. Deduced from above observation, the Hv-EREBP gene must play a role in powdery mildew resistant pathway or ethylene defense pathway in H.villosa.
Tne clone containing a full length Hv-PKI gene has a 1519bp insert, in which a 1089bp complete ORF representing a full length Hv-PKI gene was identified. The Hv-PKI gene encoded a deduced protein composed with 362 amino acids, in which a complete protein kinase domain was identified. This deduced protein has 93% identity with O.sativa protein kinase interactor (GenBank accession AAS98413). Subcellular localization analysis showed that PKI-GFP fusion proteins expressed intensively in the cytoplasm and on the membrane of onin epidermal cells, the CDS fragment from the start code to the coserved domain encoded the amino adid sequence required for accurate positioning of this protein. Expression of the fusion protein Hv-PKI was carried out with the pET expression system successfully. Recombinant plasmid was constructed by insertion of the full length Hv-PKI gene into the pET32a vector, and transformed into the competent host cell BL21(DE3). After induction by IPTG, compared with the controls, the host stains carrying the recombinant plasmid has produced a unique band whose molecular weight was equal to the deduced molecular weight of Hv-PKI protein, i.e.63.3KD. Analyzed by Real-time RT-PCR, expression of this gene was induced remarkably by exogenous hormone salicylic acid or Bgt. On the contrary, no sharp expression increase or decrease was detected in leaves of H.villosa after application with exogenous ethylene or jasmonic acid. Deduced from above observation, this gene must involve in powdery mildew reisitance and salicylic acid defense pathway in H.villosa.
To clone candidate gene for Pm21, suppression subtractive hybridization was conducted between an isogenic resistant line carrying Pm21 and its recurrent parent Yangmai 5 by Chen et al., and a resistance gene analog (RGA) Ta-LRR2 (GeneBank accession ABQ53156.1) co-segregating with Pm21 was obtained. Afterwards, a resistance gene analog polymorphic molecular marker CINAU16 (NAU/XiBao 16) linked to Pm21 was developed from Ta-LRR2 sequence. Ta-LRR2 had an increased expression level in the resistant wheat T6VS/6AL after inoculation with Bgt. In this work, a homologous RGA sequence Hv-LRR encoding 226 amino acids (GenBank accession FJ711057) was cloned, assigned on the short arm of 6V chromosome in H. villosa. Expression of the Hv-LRR was dramatically induced in leaves of H.villosa challenged by Bgt, but not induced by exogenous hormones, i.e. ethylene, salicylic acid, jasmonic acid. This gene was deduced taking part in the powdery mildew resistant pathway in H. villosa.
From our experience, tissue culture system for H.villosa was still quite difficult to be established at present time. To fast evaluate gene function, an effective and persistent virus-induced gene silencing (VIGS) system was established with barley stripe mosaic virus (BSMV) for H.villosa. Fluorescence observation of GFP gene expression showed that BSMV:GFP can deliver systemically from leaf to leaf in the early growing stage of the plant after inoculation. Characterization of phytoene desaturase (PDS) gene silencing process and Real-time RT-PCR detection showed that BSMV:PDS carrying reverse inserted fragment of PDS gene can infect H.villosa systemically, and suppress PDS transcripts as early as six days after inoculation. The photobleaching phenotype occurred mainly on the newly upper leaves, and PDS gene silencing process carried thorough the whole growing period in H.villosa. This was the first report that BSMV was used successfully for VIGS in a wild relative species of wheat. The established VIGS system will surely be a powerful reverse genetics tool for gene function study in H.villosa.
With the established VIGS system, function analysis of two genes, i.e. the Hv-EREBP gene and a serine/threonine protein kinase(Hv-S/TPK) gene, were carried out. Cloned by Cao et al. in our laboratory previously, the Hv-S/TPK gene was a candidate gene for Pm21, and located on the short arm of 6 V chromosome in H. villosa. Expression of the Hv-EREBP gene or the Hv-S/TPK gene was dramatically down-regulated in plants inoculated with BSMV:Hv-EREBP or BSMV:Hv-S/TPK. Infection percentages by Bgt increased on leaves of H. villosa in which Hv-EREBP or Hv-S/TPK were silenced, and resistance to powdery mildew decreased. All these results showed that Hv-EREBP and Hv-S/TPK definitely played a role in powdery mildew resistant pathway in H.villosa.
引文
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