抗玉米粗缩病RNA干扰载体的构建和农杆菌介导的遗传转化
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摘要
玉米粗缩病是由玉米粗缩病毒、水稻黑条矮缩病毒或Mal de Rio Cuarto virus引起的一种世界性病毒病,主要通过灰飞虱进行持久性传播。近年来,该病在我国玉米部分主产区流行,给玉米生产造成了严重的经济损失。因此,怎样控制玉米粗缩病的发病率,是一个具有重要现实意义的问题。选育和推广抗病品种是防治玉米粗缩病最经济、有效的方法。目前,粗缩病抗性育种主要是利用玉米种质资源的遗传抗性。但高抗粗缩病的种质资源非常缺乏,使得长期以来传统抗病育种工作进展缓慢,难以满足生产上对品种抗性的要求。
RNA干扰(RNAi)现象是生物进化上形成的抵御外来核酸侵犯的防御机制。研究发现,将来源于病毒的基因片段以反向重复的方式导入植物,其转录后形成的双链发夹RNA结构(hairpin RNA, hpRNA)可有效引发病毒基因沉默,使转基因植物获得对该病毒及相近病毒的抗性。因此,本研究利用RNAi原理,将引起玉米粗缩病的病毒外层衣壳蛋白基因和核心外壳蛋白基因的部分序列构建成RNAi表达载体,通过农杆菌介导法转化玉米,以期获得阳性转化植株,并使其自交纯合,为后续抗病性鉴定提供纯合的转化株系。研究结果如下：
1)构建了具有反向重复结构的中间载体pSK-303、pSK-452、pSK-291,该中间载体可为其它RNA干扰载体构建所用。首先对NCBI上提交的玉米粗缩病毒外壳蛋白(coat protein, CP)基因S10和水稻黑条矮缩病毒CP基因S10、S8进行序列同源性分析,选择保守区域中的303bp、452bp、291bp片段作为RNAi靶序列。利用两组带有酶切位点的特异引物扩增模板序列,分别正向、反向连入到pSK-int载体,构建成具有反向重复结构的中间载体。其中,正反向片段之间间隔一段玉米内含子序列,便于转录后形成发夹结构。
2)根据构建成的具有反向重复结构的中间载体pSK-303、pSK-452、pSK-291,构建成了适用于农杆菌介导的三个植物表达载体,分别为pRNAi-303、pRNAi-452、pRNAi-291。利用SpeI和XhoI限制性内切酶将中间载体上的反向重复序列切下,连接到植物表达载体pJIM19,构建成本试验的RNAi表达载体。
3)通过冻融法将表达载体质粒转入农杆菌菌株EHA105,利用芽尖法成功将植物表达载体pRNAi-303转化玉米自交系“18-599”中,获得了7个独立的阳性转化株系,T1代PCR检测获得了23个独立的阳性转化株系。其它两个表达载体也以玉米自交系“18-599”胚性愈伤组织为受体进行了遗传转化,经1.5、3、5 mg/L Basta筛选后,获得了抗性愈伤组织,目前正在分化再生植株。
Maize rough dwarf disease (MRDD) is a worldwide viral disease caused by maize rough dwarf virus (MRDV)、Rice black streaked dwarf virus (RBSDV) or Mal de Rio Cuarto virus (MRCV), and persistently transmitted by Laodelphax striatellus Fallen in a circulative manner. In recent years, the disease was epidemic in our part of the main producing areas of corn and caused serious economic losses. Therefore, how to control MRDD is an issue of great pratical significance. Screening and cultivating resistant resource is the most economic and effective way to control MRDD. Currently, maize rough dwarf disease resistance breeding mainly using genetic resistance of maize germplasm. However, high resistance to rough dwarf virus is a lack of germplasm resources. The work of resistance breeding is a long process. It is difficult to meet the production requirements through conventional breeding.
RNA interference (RNAi) is an evolutionarily conserved mechanism targeted against invasive or mobile RNA elements. It was demonstrated that silencing can be effectively triggered when invert repeat (IR) fragments deriving from pathogen was introduced in host plants, and endowed transgenic plant resistance to the virus and similar virus. Therefore, based RNAi principle, this study use part of MRDD outer coat protein gene and the core coat protein gene to construct RNAi expression vector and transform to maize by Agrobacterium-mediated in order to obtain positive transgenic plants. And make it self homozygous for the subsequent identification of disease resistance provide homozygous transgenic lines. The results are as follows:
1) The intermediate vectors pSK-303、pSK-452、pSK-291 were constructed with inverted repeats, which could be applied to other RNA interference. Firstly, sequences of MRDV CP genes S10 and RBSDV CP genes S10 and S8 submitted on NCBI were analysised, and 303bp,452bp,291bp fragment of the conserved domain were selected as RNAi target sequence. Two pairs of primer with restriction sites were used to amplify the sense and anti-sense fragments, with double enzyme digestion, the sense and anti-sense fragments were linked into intermediate vectors. In order to produce a intron-hairpin structure, there is a maize intron between sense and anti-sense fragment.
2) According constructed with inverted repeats of intermediate vector pSK-303, pSK-452, pSK-291, became applicable to build three plants expression vector by Agrobacterium-mediated. They are pRNAi-303, pRNAi-452 and pRNAi-291. Using SpeI and XhoI restriction enzyme cut inverted repeat sequence, connected to the plant expression vector pJIM19, construction RNAi expression vector.
3) The pRNAi-303 expression vector was transformed into the Agrobacterium strain EHA105 by freeze-thaw method and used to inplanta transform the apical portion of "18-599" mediated by Agrobacterium, and obtained 3 independent positive plants. PCR detection of T1 generation, obtained 23 positive independent transgenic lines. Two other expression vectors were genetic transformation. Maize inbred line "18-599" embryogenic callus was the receptor of genetic transformation. After screening with 1.5,3,5 mg/L Basta, the resistant callus are being differentiated.
RNA干扰(RNAi)现象是生物进化上形成的抵御外来核酸侵犯的防御机制。研究发现,将来源于病毒的基因片段以反向重复的方式导入植物,其转录后形成的双链发夹RNA结构(hairpin RNA, hpRNA)可有效引发病毒基因沉默,使转基因植物获得对该病毒及相近病毒的抗性。因此,本研究利用RNAi原理,将引起玉米粗缩病的病毒外层衣壳蛋白基因和核心外壳蛋白基因的部分序列构建成RNAi表达载体,通过农杆菌介导法转化玉米,以期获得阳性转化植株,并使其自交纯合,为后续抗病性鉴定提供纯合的转化株系。研究结果如下：
1)构建了具有反向重复结构的中间载体pSK-303、pSK-452、pSK-291,该中间载体可为其它RNA干扰载体构建所用。首先对NCBI上提交的玉米粗缩病毒外壳蛋白(coat protein, CP)基因S10和水稻黑条矮缩病毒CP基因S10、S8进行序列同源性分析,选择保守区域中的303bp、452bp、291bp片段作为RNAi靶序列。利用两组带有酶切位点的特异引物扩增模板序列,分别正向、反向连入到pSK-int载体,构建成具有反向重复结构的中间载体。其中,正反向片段之间间隔一段玉米内含子序列,便于转录后形成发夹结构。
2)根据构建成的具有反向重复结构的中间载体pSK-303、pSK-452、pSK-291,构建成了适用于农杆菌介导的三个植物表达载体,分别为pRNAi-303、pRNAi-452、pRNAi-291。利用SpeI和XhoI限制性内切酶将中间载体上的反向重复序列切下,连接到植物表达载体pJIM19,构建成本试验的RNAi表达载体。
3)通过冻融法将表达载体质粒转入农杆菌菌株EHA105,利用芽尖法成功将植物表达载体pRNAi-303转化玉米自交系“18-599”中,获得了7个独立的阳性转化株系,T1代PCR检测获得了23个独立的阳性转化株系。其它两个表达载体也以玉米自交系“18-599”胚性愈伤组织为受体进行了遗传转化,经1.5、3、5 mg/L Basta筛选后,获得了抗性愈伤组织,目前正在分化再生植株。
Maize rough dwarf disease (MRDD) is a worldwide viral disease caused by maize rough dwarf virus (MRDV)、Rice black streaked dwarf virus (RBSDV) or Mal de Rio Cuarto virus (MRCV), and persistently transmitted by Laodelphax striatellus Fallen in a circulative manner. In recent years, the disease was epidemic in our part of the main producing areas of corn and caused serious economic losses. Therefore, how to control MRDD is an issue of great pratical significance. Screening and cultivating resistant resource is the most economic and effective way to control MRDD. Currently, maize rough dwarf disease resistance breeding mainly using genetic resistance of maize germplasm. However, high resistance to rough dwarf virus is a lack of germplasm resources. The work of resistance breeding is a long process. It is difficult to meet the production requirements through conventional breeding.
RNA interference (RNAi) is an evolutionarily conserved mechanism targeted against invasive or mobile RNA elements. It was demonstrated that silencing can be effectively triggered when invert repeat (IR) fragments deriving from pathogen was introduced in host plants, and endowed transgenic plant resistance to the virus and similar virus. Therefore, based RNAi principle, this study use part of MRDD outer coat protein gene and the core coat protein gene to construct RNAi expression vector and transform to maize by Agrobacterium-mediated in order to obtain positive transgenic plants. And make it self homozygous for the subsequent identification of disease resistance provide homozygous transgenic lines. The results are as follows:
1) The intermediate vectors pSK-303、pSK-452、pSK-291 were constructed with inverted repeats, which could be applied to other RNA interference. Firstly, sequences of MRDV CP genes S10 and RBSDV CP genes S10 and S8 submitted on NCBI were analysised, and 303bp,452bp,291bp fragment of the conserved domain were selected as RNAi target sequence. Two pairs of primer with restriction sites were used to amplify the sense and anti-sense fragments, with double enzyme digestion, the sense and anti-sense fragments were linked into intermediate vectors. In order to produce a intron-hairpin structure, there is a maize intron between sense and anti-sense fragment.
2) According constructed with inverted repeats of intermediate vector pSK-303, pSK-452, pSK-291, became applicable to build three plants expression vector by Agrobacterium-mediated. They are pRNAi-303, pRNAi-452 and pRNAi-291. Using SpeI and XhoI restriction enzyme cut inverted repeat sequence, connected to the plant expression vector pJIM19, construction RNAi expression vector.
3) The pRNAi-303 expression vector was transformed into the Agrobacterium strain EHA105 by freeze-thaw method and used to inplanta transform the apical portion of "18-599" mediated by Agrobacterium, and obtained 3 independent positive plants. PCR detection of T1 generation, obtained 23 positive independent transgenic lines. Two other expression vectors were genetic transformation. Maize inbred line "18-599" embryogenic callus was the receptor of genetic transformation. After screening with 1.5,3,5 mg/L Basta, the resistant callus are being differentiated.
引文
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