甜瓜APX和Mlo基因的克隆与功能分析
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摘要
甜瓜是重要的瓜类作物,在世界范围内广泛栽培。近年来,甜瓜白粉病逐渐成为栽培中的主要病害,在露地以及保护地生产条件下,发病后均可导致严重减产,品质下降。常见病原菌主要为瓜单丝壳属白粉菌和二孢白粉菌,瓜单丝壳白粉菌的危害性要大于二孢白粉菌。生产上主要通过常规杂交选育的方法,培育甜瓜抗白粉病品种,但是由于白粉病原菌生理小种的演替速度很快,所以抗病育种往往落后于小种更替。施用化学杀菌剂是目前防控白粉病的主要手段,但又往往损伤了叶片和带来了污染。因此,通过基因工程,创造广谱抗白粉病的甜瓜种质具有非常重要的意义。
本研究从甜瓜中克隆到APX和Mlo家族基因,研究了各基因在白粉病胁迫条件的表达,发现APX和CmMlo家族基因在白粉病发病过程中具有一定的相关性,其中CmMlo2在白粉病发病过程中具有重要作用,可能参与了白粉病抗性机制的负调控过程,缺失Mlo会降低白粉病入侵的门槛。将CmMlo2构建了RNA干扰载体,转化甜瓜,获得了抗白粉病的To代转基因植株,实验取得的主要结果如下:
1.利用同源序列设计兼并引物,通过RT-PCR方法从甜瓜叶片中克隆到抗坏血酸过氧化物酶APX基因的中间片段,再通过5′RACE和3′RACE分别克隆得到5′端片段和3′端片段,拼接后设计特异引物扩增到全长1012bp cDNA,ORF为750bp,编码249个氨基酸的多肽,分子量大约为27.3kDa。同源序列比对表明,甜瓜APX基因和黄瓜、番茄、玉米、小麦、大麦、拟南芥等APX基因高度同源,将该基因命名为CmAPX,GenBank登录号:EF693949。根据获得的cDNA序列,设计引物,从基因组扩增得到甜瓜APX的gDNA序列,与cDNA序列比对发现CmAPX包含10个外显子,9个内含子,所有内含子均符合GT-AG剪切规则。
2.构建了pET24-CmAPX原核表达载体,诱导1、2、3、4、5h后蛋白表达量分别占总融合蛋白的40%、47.5%、50.6%、51.7%、51.6%。表明CmAPX基因在pET24a(+)载体中获得高效表达。表达产物具有APX酶活性,说明CmAPX编码抗坏血酸过氧化物酶基因。
3.通过RT-PCR半定量,甜瓜APX基因在不同器官中呈非特异性表达,在叶片中的mRNA含量明显高于根、茎,花及果实。经白粉病诱导0、1、3、5、7d后,表达量在5d时达到最高值,和叶片中酶活性变化一致,表明CmAPX可能参与了白粉病生物胁迫过程。
4.根据大麦、拟南芥等Mlo基因的保守序列,设计兼并引物,扩增得到了3个甜瓜Mlo类似中间片段,进一步通过Race技术,获得了3个cDNA全长,分别命名为CmMlo1,CmMlo2和CmMlo3。其中CmMlo1全长1551bp,编码516个氨基酸;CmMlo2全长1713bp,编码570个氨基酸;CmMlo3全长1464bp,编码487个氨基酸序列比对结果表明,CmMlo属于基因家族,和拟南芥的Mlo基因同源性在70%左右。定量和半定量分析结果表明,CmMlo在甜瓜组织中呈特异性表达,在受到白粉病胁迫条件下,CmMlo2表达量上调,CmMlo2可能参与了白粉病发病的负调控过程。
5.跨膜蛋白预测表明,CmMlo2为跨膜蛋白。具有7个跨膜螺旋结构。构建pROK-CmMlo2-GFP融合蛋白并转化洋葱表皮细胞,结果表明,CmMlo2蛋白定位于细胞膜。将pROK-CmMlo2-GFP融合蛋白转化烟草,野生型烟草出现了荧光,构建发夹结构pFGC1008-CmMlo2干扰载体,转入荧光烟草可以导致荧光消失,表明干扰载体对靶序列是有效的。
6.建立了稳定高效的甜瓜再生体系,优化了甜瓜遗传转化体系,通过农杆菌介导法转化感白粉病甜瓜材料,获得了转化植株。PCR结果及荧光定量分析表明,pFGC1008-CmMlo2已经整合到甜瓜基因组,接种白粉病鉴定表明,转化植株具有白粉病抗性,叶片表面未见白粉病菌丝生长。转化植株出现卷须早,长势较对照(野生型植株)弱,叶片大小、色泽没有明显区别,RNAi植株没有出现明显的生理缺陷。
Melon (Cucumis melon L.) is a valuable cash crop grown throughout the word. It is a member of the genus Cucumis, in the family Cucurbitaceae. Powdery mildew is one of the most important limiting factors for cucurbits production. Powdery mildew is a severe disease which causes the reduction of melon production in worldwide. As the most characteristic visual symptom the disease induces development of a whitish, talcum-like powdery growth on stems, petioles and leaf surfaces. Infected leaves usually wither and die, and plants senesce prematurely. It frequently causes significant yield losses and reduction of fruit quality in agricultural settings, including field farming and greenhouse. It has been suggested that recombination may play an important role in the virulence variation of P. xanthii in cucurbits. So it is difficulty to develop a normal breeding method to obtain melon cultivars resistance to different fungal races. Therefore, through genetic engineering to obtain the durable, broad-spectrum resistance variant of melon is very important.
We successfully cloned the APX and Mlo family genes form muskmelon. The analysis showed that these genes maybe related with occurrence of the powdery mildew. The result of RT -PCR and Real-time PCR indicate that the CmMlo2 functions as a negative regulator that suppressed plant defenses in uninfected tissues. Loss of MLO reduces the threshold at which cells respond defensively to powdery mildew infections. We obtained the translated melon plant after constructed RNAi vector.
1. The RT PCR and RACE methods were used to obtain the cDNA sequence of an ascorbate peroxidase (APX) gene after the leaf was induced with powdery mildew. A pair of primers was designed for obtaining the cDNA and genomic DNA sequence. The cDNA length of APX gene is 1047bp with a 750bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns .The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74-97%.
2. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E.coli. This is the first report on cloning and sequencing the APX gene from melon.
3. For determining the function of CmAPX we inoculated the leaves with powdery mildew, moderate accumulation of CmAPX mRNA was observed in initial leaves, and then increased during infection process. In the melon leaves the mRNA level sharply increased after 3d,and remained high to 5d, after 7d the mRNA level reached to normal level. We also detected the APX activity of the melon leaves. The results showed that the APX activity was increased during the leaves was induced with powdery mildew .The activity reached a maximum level after the leaves induced within 5d. These results suggested that CmAPX involved in the infection process of powdery mildew.
4. The RT-PCR and RACE methods were used to obtain the cDNA sequence of three Mlo genes of muskmelon after the leaves were induced with powdery mildew. The three Mlo genes of melon were designated CmMlo1 (GeneBank Acc: FJ713541), CmMlo2 (GeneBank Acc: FJ713542) and CmMlo3 (GeneBank Acc: FJ713543). CmMlo1 was 1551 bp in length and contained a putative ORF encoding 516 amino acid residues, while CmMlo2 cDNA was 1713 bp in length and contained a putative ORF encoding 570 amino acid residues. CmMlo3 was 1464 bp in length and contained a putative ORF encoding 487 amino acid residues. To detect similarities and differences in individual amino acid sequence positions, we aligned the deduced amino acid sequence of CmMlo with the sequence of other plant Mlo in the database of NCBI Blast search. The comparison of identities among the homologues isolated in this study indicated that CmMlo1, CmMlo2 and CmMlo3 encode polypeptides that are highly sequence related to barley Mlo, Arabidopsis AtMLO2, AtMLO6, and AtMLO12. Semi-quantitative RT-PCR and Real-time PCR indicated Cmmlo2 may play a important role in occurrence of powdery mildew.
5. Predicted topology of Cmmlo2 and assays of pROK-Cmmlo2-GFP fusion protein in onion showed Cmmlo2 is an integral plasma membrane-localized protein.
6. After The efficient of dsRNA constructs were examined via Cmmlo2-GFP fusion protein and pFGC1008-Cmmlo2 were Co-transformation in Nicotiana benthamiana the dsRNA constructs were introduced into Agrobacterium tumefaciens and transformed into melon wild type with the methods of Agrobacterium-mediated leaf disc transformation. The material of broad-spectrum powdery mildew resistance against P. xanthii in muskmelon was obtained by ihpRNAi-mediated knock-down of Cmmlo2.
本研究从甜瓜中克隆到APX和Mlo家族基因,研究了各基因在白粉病胁迫条件的表达,发现APX和CmMlo家族基因在白粉病发病过程中具有一定的相关性,其中CmMlo2在白粉病发病过程中具有重要作用,可能参与了白粉病抗性机制的负调控过程,缺失Mlo会降低白粉病入侵的门槛。将CmMlo2构建了RNA干扰载体,转化甜瓜,获得了抗白粉病的To代转基因植株,实验取得的主要结果如下:
1.利用同源序列设计兼并引物,通过RT-PCR方法从甜瓜叶片中克隆到抗坏血酸过氧化物酶APX基因的中间片段,再通过5′RACE和3′RACE分别克隆得到5′端片段和3′端片段,拼接后设计特异引物扩增到全长1012bp cDNA,ORF为750bp,编码249个氨基酸的多肽,分子量大约为27.3kDa。同源序列比对表明,甜瓜APX基因和黄瓜、番茄、玉米、小麦、大麦、拟南芥等APX基因高度同源,将该基因命名为CmAPX,GenBank登录号:EF693949。根据获得的cDNA序列,设计引物,从基因组扩增得到甜瓜APX的gDNA序列,与cDNA序列比对发现CmAPX包含10个外显子,9个内含子,所有内含子均符合GT-AG剪切规则。
2.构建了pET24-CmAPX原核表达载体,诱导1、2、3、4、5h后蛋白表达量分别占总融合蛋白的40%、47.5%、50.6%、51.7%、51.6%。表明CmAPX基因在pET24a(+)载体中获得高效表达。表达产物具有APX酶活性,说明CmAPX编码抗坏血酸过氧化物酶基因。
3.通过RT-PCR半定量,甜瓜APX基因在不同器官中呈非特异性表达,在叶片中的mRNA含量明显高于根、茎,花及果实。经白粉病诱导0、1、3、5、7d后,表达量在5d时达到最高值,和叶片中酶活性变化一致,表明CmAPX可能参与了白粉病生物胁迫过程。
4.根据大麦、拟南芥等Mlo基因的保守序列,设计兼并引物,扩增得到了3个甜瓜Mlo类似中间片段,进一步通过Race技术,获得了3个cDNA全长,分别命名为CmMlo1,CmMlo2和CmMlo3。其中CmMlo1全长1551bp,编码516个氨基酸;CmMlo2全长1713bp,编码570个氨基酸;CmMlo3全长1464bp,编码487个氨基酸序列比对结果表明,CmMlo属于基因家族,和拟南芥的Mlo基因同源性在70%左右。定量和半定量分析结果表明,CmMlo在甜瓜组织中呈特异性表达,在受到白粉病胁迫条件下,CmMlo2表达量上调,CmMlo2可能参与了白粉病发病的负调控过程。
5.跨膜蛋白预测表明,CmMlo2为跨膜蛋白。具有7个跨膜螺旋结构。构建pROK-CmMlo2-GFP融合蛋白并转化洋葱表皮细胞,结果表明,CmMlo2蛋白定位于细胞膜。将pROK-CmMlo2-GFP融合蛋白转化烟草,野生型烟草出现了荧光,构建发夹结构pFGC1008-CmMlo2干扰载体,转入荧光烟草可以导致荧光消失,表明干扰载体对靶序列是有效的。
6.建立了稳定高效的甜瓜再生体系,优化了甜瓜遗传转化体系,通过农杆菌介导法转化感白粉病甜瓜材料,获得了转化植株。PCR结果及荧光定量分析表明,pFGC1008-CmMlo2已经整合到甜瓜基因组,接种白粉病鉴定表明,转化植株具有白粉病抗性,叶片表面未见白粉病菌丝生长。转化植株出现卷须早,长势较对照(野生型植株)弱,叶片大小、色泽没有明显区别,RNAi植株没有出现明显的生理缺陷。
Melon (Cucumis melon L.) is a valuable cash crop grown throughout the word. It is a member of the genus Cucumis, in the family Cucurbitaceae. Powdery mildew is one of the most important limiting factors for cucurbits production. Powdery mildew is a severe disease which causes the reduction of melon production in worldwide. As the most characteristic visual symptom the disease induces development of a whitish, talcum-like powdery growth on stems, petioles and leaf surfaces. Infected leaves usually wither and die, and plants senesce prematurely. It frequently causes significant yield losses and reduction of fruit quality in agricultural settings, including field farming and greenhouse. It has been suggested that recombination may play an important role in the virulence variation of P. xanthii in cucurbits. So it is difficulty to develop a normal breeding method to obtain melon cultivars resistance to different fungal races. Therefore, through genetic engineering to obtain the durable, broad-spectrum resistance variant of melon is very important.
We successfully cloned the APX and Mlo family genes form muskmelon. The analysis showed that these genes maybe related with occurrence of the powdery mildew. The result of RT -PCR and Real-time PCR indicate that the CmMlo2 functions as a negative regulator that suppressed plant defenses in uninfected tissues. Loss of MLO reduces the threshold at which cells respond defensively to powdery mildew infections. We obtained the translated melon plant after constructed RNAi vector.
1. The RT PCR and RACE methods were used to obtain the cDNA sequence of an ascorbate peroxidase (APX) gene after the leaf was induced with powdery mildew. A pair of primers was designed for obtaining the cDNA and genomic DNA sequence. The cDNA length of APX gene is 1047bp with a 750bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns .The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74-97%.
2. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E.coli. This is the first report on cloning and sequencing the APX gene from melon.
3. For determining the function of CmAPX we inoculated the leaves with powdery mildew, moderate accumulation of CmAPX mRNA was observed in initial leaves, and then increased during infection process. In the melon leaves the mRNA level sharply increased after 3d,and remained high to 5d, after 7d the mRNA level reached to normal level. We also detected the APX activity of the melon leaves. The results showed that the APX activity was increased during the leaves was induced with powdery mildew .The activity reached a maximum level after the leaves induced within 5d. These results suggested that CmAPX involved in the infection process of powdery mildew.
4. The RT-PCR and RACE methods were used to obtain the cDNA sequence of three Mlo genes of muskmelon after the leaves were induced with powdery mildew. The three Mlo genes of melon were designated CmMlo1 (GeneBank Acc: FJ713541), CmMlo2 (GeneBank Acc: FJ713542) and CmMlo3 (GeneBank Acc: FJ713543). CmMlo1 was 1551 bp in length and contained a putative ORF encoding 516 amino acid residues, while CmMlo2 cDNA was 1713 bp in length and contained a putative ORF encoding 570 amino acid residues. CmMlo3 was 1464 bp in length and contained a putative ORF encoding 487 amino acid residues. To detect similarities and differences in individual amino acid sequence positions, we aligned the deduced amino acid sequence of CmMlo with the sequence of other plant Mlo in the database of NCBI Blast search. The comparison of identities among the homologues isolated in this study indicated that CmMlo1, CmMlo2 and CmMlo3 encode polypeptides that are highly sequence related to barley Mlo, Arabidopsis AtMLO2, AtMLO6, and AtMLO12. Semi-quantitative RT-PCR and Real-time PCR indicated Cmmlo2 may play a important role in occurrence of powdery mildew.
5. Predicted topology of Cmmlo2 and assays of pROK-Cmmlo2-GFP fusion protein in onion showed Cmmlo2 is an integral plasma membrane-localized protein.
6. After The efficient of dsRNA constructs were examined via Cmmlo2-GFP fusion protein and pFGC1008-Cmmlo2 were Co-transformation in Nicotiana benthamiana the dsRNA constructs were introduced into Agrobacterium tumefaciens and transformed into melon wild type with the methods of Agrobacterium-mediated leaf disc transformation. The material of broad-spectrum powdery mildew resistance against P. xanthii in muskmelon was obtained by ihpRNAi-mediated knock-down of Cmmlo2.
引文
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