黄瓜抗根结线虫基因工程的初步研究
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摘要
黄瓜是我国重要的蔬菜作物。近年来,随着我国种植业结构的调整,黄瓜的保护地种植和大棚种植面积迅速增加,而重茬种植和温室大棚内人工控制的环境使黄瓜生产中根结线虫的危害日趋严重,根结线虫病已成为黄瓜生产中一种毁灭性的病虫害。引起黄瓜根结线虫病的线虫主要有4个种,即南方根结线虫(Meloidogyne incognite)、爪哇根结线虫(M. javanica)、花生根结线虫(M. arenaria)和北方根结线虫(M. hapla)。黄瓜多抗自交系NC-46来源于甜瓜属的野生资源LJ90430,NC-46表现出抗爪哇根结线虫(M. javanica)和花生根结线虫生理小种1(M.arenaria race 1)以及生理小种2(M. arenaria race 2),并且还具有抗炭疽病(Anthracnose)和抗黑星病(Scab)等性状。
本研究利用基因工程技术,构建了黄瓜多抗自交系NC-46转基因毛状根的cDNA文库,并且进行了初步分析；同时,建立了利用黄瓜苗和转基因毛状根繁殖根结线虫的新方法以及一种快速分析与根相关的功能和基因的新方法,主要研究结果如下：
1.利用发根农杆菌K599(带有内源pRi2659质粒和外源pBIN-35S-GFP质粒)介导,以NC-46离体子叶为外植体,高效诱导出转基因毛状根(诱导率达76.7%)；并在此基础上成功构建了转基因毛状根的cDNA文库。构建的cDNA文库重组率为98.3%,原始库容量达2.02×106cfu；其中,基因的完整性比率为47.6%,unigene比例为66.7%。本研究不仅为利用构建的cDNA文库筛选黄瓜抗根结线虫等抗性基因提供了基础,而且为毛状根发生相关基因的克隆和毛状根发生的分子机理研究提供了前提材料。
2.利用黄瓜实生苗为宿主,在透明的玻璃瓶中成功繁殖了南方根结线虫、花生根结线虫和爪哇根结线虫,为有效分离根结线虫提供了一条新途径。此外,利用黄瓜转基因毛状根成功繁殖了南方根结线虫。上述方法不仅丰富了繁殖根结线虫的宿主,而且便于随时观察线虫侵染根形成根结的动态过程,也避免了在繁殖过程可能造成线虫逃逸对周围土壤的污染问题。
3.利用质粒pGFP和pRI101-AN构建了植物转基因表达载体pRI101-AN-gfp,将含有目标基因的重组质粒转入发根农杆菌K599中,带有重组质粒的发根农杆菌K599侵染黄瓜活体苗子叶下方0.2-0.5 cm处,诱导形成转基因毛状根,待毛状根生长至5-10 cm足以支撑植物生长时剪掉主根,即获得具有转基因毛状根的复合型黄瓜植株。利用这种复合型的黄瓜植株可以直接对黄瓜根相关功能的候选基因进行快速鉴定,无需获得转基因植株再进行鉴定,大大减少实验的操作程序,克服转基因再生效率低的缺陷。
本研究获得的结果为从黄瓜多抗自交系NC-46中克隆抗根结线虫候选基因并进行基因功能的快速有效的鉴定提供了重要基础。
Cucumber (Cucumis sativus) is the mainly vegetable in China. With the adjustment of planting method, the root-knot nematodes(Meloidogyne spp.) have become the major problem in cucumber production. The cucumber mainly infected by four nematodes of Meloidogyne incognite, M. javanica, M. arenaria and M. hapla. Several sources of resistance to root-knot nematode have been identified in wild cucumber sources. The cucumber inbred NC-46 was developed from the improved North Calinahardwickii 1(NCH 1) cucumber popularion and had LJ90430 germplasm in its background. NC-46 habours resistence to M. javanica 1、M. arenaria race 2 and M. arenaria race 2.
In this study, a cDNA library of NC-46 transgenic hairy root was constructed and analysis. A novel method for propagation root-knot nematode using cucmber plantlets and hairy root were established. And a new method of studing root gene function using composite cucumber plantlet with hairy root was developed. The results are as follows:
1.The transgenic hairy roots of NC-46 were induced from cotyledon by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 76.7%. Besides, cDNA library of the transgenic hairy roots was constructed successfully. The percentage of recombinant clones in the cDNA library was 98.3%. The original storage capacity reached 2.02×106 cfu, while the ratio of full-length gene and unigene were 47.6% and 66.7% respectively. The cDNA library is useful for screening resistant genes, and cloning of developing gene and founding the developing mechanism of hair roots.
2. Root-knot nematode of Meloidogyne incognite, M. javanica and M. arenaria can host in cucumber seedlings for propagation in transparent glass bottles. In addition, the Meloidogyne incognite could grow on hairy roots normally. It is a novel method for efficient separation of root-knot nematode. This method not only enriches the host of root-knot nematode reproduction, but also is convenient to observe the root-knot formation and prevent nematode to escape to the surrounding soil.
3. A vector pRI 101-AN-gfp was constructed with plasmids of pRI 101-AN and pGFP. After the cucumber plantlets infected by K599 with the recombinant plasmid pRI101-AN-gfp at 0.2-0.5 cm location below coteledon, the hairy root was induced. The comopsite plant was developted when the hairy root grown to length 5-10 cm. The comoposite plant could be used to study the root gene function. The utility of the procedure made possible analyses in wide gene function rapidly and easily.
The above-results provide the important foundation for cloning the candidate resistant gene to nematode from cucumber inbred line NC-46 and identification gene function rapidly and effectively.
本研究利用基因工程技术,构建了黄瓜多抗自交系NC-46转基因毛状根的cDNA文库,并且进行了初步分析；同时,建立了利用黄瓜苗和转基因毛状根繁殖根结线虫的新方法以及一种快速分析与根相关的功能和基因的新方法,主要研究结果如下：
1.利用发根农杆菌K599(带有内源pRi2659质粒和外源pBIN-35S-GFP质粒)介导,以NC-46离体子叶为外植体,高效诱导出转基因毛状根(诱导率达76.7%)；并在此基础上成功构建了转基因毛状根的cDNA文库。构建的cDNA文库重组率为98.3%,原始库容量达2.02×106cfu；其中,基因的完整性比率为47.6%,unigene比例为66.7%。本研究不仅为利用构建的cDNA文库筛选黄瓜抗根结线虫等抗性基因提供了基础,而且为毛状根发生相关基因的克隆和毛状根发生的分子机理研究提供了前提材料。
2.利用黄瓜实生苗为宿主,在透明的玻璃瓶中成功繁殖了南方根结线虫、花生根结线虫和爪哇根结线虫,为有效分离根结线虫提供了一条新途径。此外,利用黄瓜转基因毛状根成功繁殖了南方根结线虫。上述方法不仅丰富了繁殖根结线虫的宿主,而且便于随时观察线虫侵染根形成根结的动态过程,也避免了在繁殖过程可能造成线虫逃逸对周围土壤的污染问题。
3.利用质粒pGFP和pRI101-AN构建了植物转基因表达载体pRI101-AN-gfp,将含有目标基因的重组质粒转入发根农杆菌K599中,带有重组质粒的发根农杆菌K599侵染黄瓜活体苗子叶下方0.2-0.5 cm处,诱导形成转基因毛状根,待毛状根生长至5-10 cm足以支撑植物生长时剪掉主根,即获得具有转基因毛状根的复合型黄瓜植株。利用这种复合型的黄瓜植株可以直接对黄瓜根相关功能的候选基因进行快速鉴定,无需获得转基因植株再进行鉴定,大大减少实验的操作程序,克服转基因再生效率低的缺陷。
本研究获得的结果为从黄瓜多抗自交系NC-46中克隆抗根结线虫候选基因并进行基因功能的快速有效的鉴定提供了重要基础。
Cucumber (Cucumis sativus) is the mainly vegetable in China. With the adjustment of planting method, the root-knot nematodes(Meloidogyne spp.) have become the major problem in cucumber production. The cucumber mainly infected by four nematodes of Meloidogyne incognite, M. javanica, M. arenaria and M. hapla. Several sources of resistance to root-knot nematode have been identified in wild cucumber sources. The cucumber inbred NC-46 was developed from the improved North Calinahardwickii 1(NCH 1) cucumber popularion and had LJ90430 germplasm in its background. NC-46 habours resistence to M. javanica 1、M. arenaria race 2 and M. arenaria race 2.
In this study, a cDNA library of NC-46 transgenic hairy root was constructed and analysis. A novel method for propagation root-knot nematode using cucmber plantlets and hairy root were established. And a new method of studing root gene function using composite cucumber plantlet with hairy root was developed. The results are as follows:
1.The transgenic hairy roots of NC-46 were induced from cotyledon by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 76.7%. Besides, cDNA library of the transgenic hairy roots was constructed successfully. The percentage of recombinant clones in the cDNA library was 98.3%. The original storage capacity reached 2.02×106 cfu, while the ratio of full-length gene and unigene were 47.6% and 66.7% respectively. The cDNA library is useful for screening resistant genes, and cloning of developing gene and founding the developing mechanism of hair roots.
2. Root-knot nematode of Meloidogyne incognite, M. javanica and M. arenaria can host in cucumber seedlings for propagation in transparent glass bottles. In addition, the Meloidogyne incognite could grow on hairy roots normally. It is a novel method for efficient separation of root-knot nematode. This method not only enriches the host of root-knot nematode reproduction, but also is convenient to observe the root-knot formation and prevent nematode to escape to the surrounding soil.
3. A vector pRI 101-AN-gfp was constructed with plasmids of pRI 101-AN and pGFP. After the cucumber plantlets infected by K599 with the recombinant plasmid pRI101-AN-gfp at 0.2-0.5 cm location below coteledon, the hairy root was induced. The comopsite plant was developted when the hairy root grown to length 5-10 cm. The comoposite plant could be used to study the root gene function. The utility of the procedure made possible analyses in wide gene function rapidly and easily.
The above-results provide the important foundation for cloning the candidate resistant gene to nematode from cucumber inbred line NC-46 and identification gene function rapidly and effectively.
引文
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