一个玉米RING蛋白基因克隆和功能分析
摘要
产量性状在作物育种中起着十分重要的作用,本研究从玉米中克隆了一个水稻产量性状主效基因GW2的同源基因,命名为ZmGW2。利用生物信息学分析该基因的初步结构和功能;半定量RT-PCR检测ZmGW2在不同组织、不同处理下的表达情况;采用农杆菌介导的转基因技术,将ZmGW2导入拟南芥以及玉米中。主要结果如下:
1.生物信息学分析,推测ZmGW2蛋白含有431个氨基酸,且也具有RING指结构。所有的RING指蛋白都如E3泛素蛋白质连接酶,参与一系列生物学领域。
2.半定量RT-PCR分析表明,ZmGW2可以被高盐(NaCl)和脱落酸(ABA)诱导,且具有抵抗干旱和冷害的能力。另外,在不同的组织中,ZmGW2的表达模式也不一样,在茎和子粒中相对较高,而且在子代品种的花丝中ZmGW2的表达量比亲本高。这些结果表明,ZmGW2可能在产量方面、植物逆境反应及生殖生长发育过程中起多重作用。
3.构建ZmGW2过表达载体和AtGW2 RNA干扰植物转化载体并转化拟南芥,获得的转基因pSuperCAMBIA1300(+)ihpAtGW2后代植株的子粒比转基因pSuperCAMBIA1300(+)ZmGW2的更大,千粒重相对也高,这表明ZmGW2、AtGW2负调控拟南芥子粒大小及粒重。
4.以野生型拟南芥作对照,对转基因pSuperCAMBIA1300(+)ZmGW2拟南芥T1代进行干旱(土壤含水量36%)处理、低温(-6℃)处理以及NaCl、ABA不同浓度下的诱导,结果显示转基因pSuperCAMBIA1300(+)ZmGW2拟南芥幼苗的生长情况均好于野生型拟南芥。这表明ZmGW2参与了逆境胁迫响应。
High yield play important roles in breeding. In this study, a gene was cloned from maize (Zea mays L.) by RT-PCR method and named as ZmGW2. The initial structure and function of ZmGW2 was analyzed by bioinformatics.The expression of ZmGW2 was detected by sqRT-PCR in different tissues and different treatments. ZmGW2 was introduced into maize and Arabidopsis thaliana by Agrobacterium-mediated transformation.
1. Bioinformatics analysis revealed that the predicted ZmGW2 protein has 431 amino acids, and has RING-type domain. all RING proteins act as E3 ubiquitin protein ligases, with implications for a variety of biological areas.
2. SqRT-PCR analysis showed that the ZmGW2 expression was induced by salt (NaCl) and abscisic acid (ABA), and ZmGW2 was resistant to drought and cold. Furthermore, the ZmGW2 showed different expression patterns in different tissues and was more abundant in stems and kernels. ZmGW2 expression was higher in silk of F1 than parents. These results suggest that may play multiple roles in yield, plant stress response, as well as reproductive growth and development.
3. Over-expression vector of ZmGW2 and RNA interference expression vector of AtGW2 were constructed and transformed into Arabidopsis thaliana.The grain size and kilo kernels weight of ZmGW2 overexpression transgenic plants were lower than those of AtGW2 RNA interference transgenic plants. These results suggested that ZmGW2 and AtGW2 negatively regulates the grain size and weight in Arabidopsis.
4. Compared with wild-type Arabidopsis, T1 seeds and plants of ZmGW2 overexpression transgenic plants were treated with drought, cold, NaCl and ABA. The growth of transgenic Arabidopsis was all better than wild-type plants. The results showed that ZmGW2 may play multiple roles in plant stress response.
1.生物信息学分析,推测ZmGW2蛋白含有431个氨基酸,且也具有RING指结构。所有的RING指蛋白都如E3泛素蛋白质连接酶,参与一系列生物学领域。
2.半定量RT-PCR分析表明,ZmGW2可以被高盐(NaCl)和脱落酸(ABA)诱导,且具有抵抗干旱和冷害的能力。另外,在不同的组织中,ZmGW2的表达模式也不一样,在茎和子粒中相对较高,而且在子代品种的花丝中ZmGW2的表达量比亲本高。这些结果表明,ZmGW2可能在产量方面、植物逆境反应及生殖生长发育过程中起多重作用。
3.构建ZmGW2过表达载体和AtGW2 RNA干扰植物转化载体并转化拟南芥,获得的转基因pSuperCAMBIA1300(+)ihpAtGW2后代植株的子粒比转基因pSuperCAMBIA1300(+)ZmGW2的更大,千粒重相对也高,这表明ZmGW2、AtGW2负调控拟南芥子粒大小及粒重。
4.以野生型拟南芥作对照,对转基因pSuperCAMBIA1300(+)ZmGW2拟南芥T1代进行干旱(土壤含水量36%)处理、低温(-6℃)处理以及NaCl、ABA不同浓度下的诱导,结果显示转基因pSuperCAMBIA1300(+)ZmGW2拟南芥幼苗的生长情况均好于野生型拟南芥。这表明ZmGW2参与了逆境胁迫响应。
High yield play important roles in breeding. In this study, a gene was cloned from maize (Zea mays L.) by RT-PCR method and named as ZmGW2. The initial structure and function of ZmGW2 was analyzed by bioinformatics.The expression of ZmGW2 was detected by sqRT-PCR in different tissues and different treatments. ZmGW2 was introduced into maize and Arabidopsis thaliana by Agrobacterium-mediated transformation.
1. Bioinformatics analysis revealed that the predicted ZmGW2 protein has 431 amino acids, and has RING-type domain. all RING proteins act as E3 ubiquitin protein ligases, with implications for a variety of biological areas.
2. SqRT-PCR analysis showed that the ZmGW2 expression was induced by salt (NaCl) and abscisic acid (ABA), and ZmGW2 was resistant to drought and cold. Furthermore, the ZmGW2 showed different expression patterns in different tissues and was more abundant in stems and kernels. ZmGW2 expression was higher in silk of F1 than parents. These results suggest that may play multiple roles in yield, plant stress response, as well as reproductive growth and development.
3. Over-expression vector of ZmGW2 and RNA interference expression vector of AtGW2 were constructed and transformed into Arabidopsis thaliana.The grain size and kilo kernels weight of ZmGW2 overexpression transgenic plants were lower than those of AtGW2 RNA interference transgenic plants. These results suggested that ZmGW2 and AtGW2 negatively regulates the grain size and weight in Arabidopsis.
4. Compared with wild-type Arabidopsis, T1 seeds and plants of ZmGW2 overexpression transgenic plants were treated with drought, cold, NaCl and ABA. The growth of transgenic Arabidopsis was all better than wild-type plants. The results showed that ZmGW2 may play multiple roles in plant stress response.
引文
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