摘要
蚜虫属同翅目昆虫，种类繁多，侵害目标广泛。它通过口器深入植物的韧皮部吸收营养，并携带和传播病毒，造成农作物的产量和品质的下降。植物抗虫基因工程为控制害虫的危害提供了新的途径。随着越来越多的抗虫基因被导入植物以提高植物的抗虫能力，人们发现：寻找新的高效抗虫基因，仍是现阶段抗虫基因工程的关键；对植物抗虫能力的提高不能单单依靠某个抗虫基因的转入就能实现；抗虫基因在转基因植物体内表达不稳定甚至出现转基因沉默；等等。基因融合可以使多个抗虫基因协同作用，提高植物的抗虫能力和抗虫谱并实现基因同步表达，并以完全独立的不同的杀虫蛋白的形式发挥活性，因此在植物抗虫基因工程中有很高的利用价值。此外，利用MARs序列(Matrix attachment regios，MARs)是在染色体水平克服外源基因失活的一种有效策略。
本研究采用同源扩增PCR技术，从苋属的野苋菜中分离和克隆新的苋菜凝集素家族基因，并对这个基因的结构与功能进行研究，阐明在现代分子遗传育种中引入该基因的可能途径和重要意义。通过构建杀虫融合蛋白基因，即将两个抗虫基因编码序列通过接头连接在一起，构成可同时表达两种抗虫活性的融合蛋白，以期能延缓昆虫抗性的发生，而且也增强了杀虫活性。另外，在减少基因沉默的措施上，在目的基因的两侧增加基质结合序列(MARs)，以增强基因的表达能力。本研究取得的主要结果如下：
1．利用同源克隆的方法从野苋菜Amaranthus viridis L．中克隆到野苋菜凝集素基因(Amaranthus viridis aggllutinin，AVA)。转基因烟草植株的抗蚜分析表明，表达野苋莱凝集素的转基因烟草植株都不同程度地抑制了蚜虫群体发展的速度，转AVA和AVAc基因植株平均抑蚜率分别约为60.181％和50.63％。转含有内含子的AVA基因比不含内含子的编码区基因AVAc有更好的抗蚜性。AVA基因是苋菜凝集素基因家族的新成员，是一个有应用前景的新的抗虫基因。
2．本研究从三个不同品种的豌豆中分离到3个具有MARs序列特征的DNA片段MMAR．DMAR，HMAR。利用GenBank同源性搜寻发现它们是新的未注册的DNA序列。经转化烟草对GUS活性测定发现，两侧顺式重复连接MAR对外源基因表达的增强作用很明显，MMAR、HMAR和DMAR都不同程度的提高了GUS的表达，分别为载体对照植株的4.16、3.66和2.08倍。但不同个体间表达水平差异仍然比较明显。
3．通过构建MAR-AVA-MAR植物表达载体，利用农杆菌介导法转化白菜，获得抗蚜转化植株。含有AVA基因的白菜植株对桃蚜的群体发展有一定的抑制作用，在接种12天后的平均抑制桃蚜密度达55.8％；在转基因抗性植株上观察到有少量桃蚜若虫死亡的现象。无MAR的转基因植株平均桃蚜抑制率为34.3％。但含MAR的转基因单株之间基因表达也仍存在一定的差异。从总体上来说含有MAR的转基因白菜植株较不含MAR的转化植株对桃蚜具有更明显的抑制作用，且抗性程度与AVA基因的表达量呈正相关。
4．根据豇豆胰蛋白酶抑制剂CpTl和野苋菜凝集素AVAc的抗虫特性，利用豌豆MMAR，成功构建了pC2MAR-AVAc／CpTI植物表达载体。以甘蔗ROC25胚性愈伤组织为转化受体材料，建立了一个高效而又简便的农杆菌介导的甘蔗遗传转化体系，成功地实现了MAR序列介导杀虫融合基因(AVAc+CpTI)在甘蔗中转化。
Sap-sucking insects belong to Homoptera, which have piercing and suckingmouthparts and feed upon the sap of plants, and most of them are serious pests ofagricultural and horticultural crops including aphids, whiteflies, planthoppers andleafhoppers. Crop is destroyed not only by pest feeding, but also by plant viruses producedthrough lesions that the mouthparts make. Plant resistance to insects through geneticengineering provides a new approach for control pests. With more and more insect-resistant genes were found and applied, people found that to search the highinsect-resistant genes are still the key task in transgenic researches. And the enhancedability of insect-resistance of transgenics does not depend on the introduction of singleinsect-resistant gene. Gene silence and instable gene expressions are also observed ingenetic engineering. Fusion gene, which can enhance the insect-resistant ability and kindsof insects by the means of fusion of several insect-resistant genes, exerts the insect-resistantability through the respective proteins. Furthermore, application of Matrix attachmentregios is also considered an efficient strategy to hurdle the gene silence at the level ofchromosome. The objectives of the present study are to clone new aphid-resitstant genesand improve the gene express level in transgenic plants. In the present study, Amaranthusviridis aggllutinin (AVA) was cloned by PCR from Amaranthus viridis L. In order toilluminate the possiblle approaches and important signification of gene introductiontocrops, the structure and functions of AVA were analysed and the insect-resistant fusion genewas constructed by connecting the coding sequences of two resistant genes with linker,which could kept the respeetine activity and enhance the insect resistance of the transgenics, and furthermore delay the occurance of non-resistance to insects. To avoid the gene silenceand enhance gene expression, the gene was flanked on both sides by MARs in the samedirection. The main results were as follows:
1. Using total DNA isolated from Amaranthus viridis L., Amaranthus viridis agglutinin(AVA) was amplified by PCR and cloned. Sequence analysis results showed that AVA geneis consisted of 1831bp base pairs, which include one 922bp intron and two exons of 212bpand 697bp, respectively. After inverse PCR amplification, the coding region of AVA genewas obtained. The AVA gene fragments with and without introns, were inserteddownstream of 35S promoter in the vector pBI121 resulting the construction of two plantexpression vectors pBI121AVA and pBI121AVAc respectively. Leaf explants of Nicotinanatabatum var. SR1 were transformed with A. tumefaciens EHA105 harboring the expressionvector. The results of PCR and GUS detection showed that AVA gene was integrated intothe genome of the transformed tobacco plants, and indicated that AVA was expressed in thetransgenic plants. The results from insect bioassay with peach aphid (Myzus persicae)showed that the transgenic plants of pBI121AVA and pBI121AVAc were aphid resistant,evidenced by 50.63%～60.81% reduction in insect population density, or even higher than97% in some plants. The AVA gene of aphid resistance is regarded as a new member inAmaranthus lcctin families.
2. Three new MAR segments (MMAR, DMAR and MAR) were cloned from threekinds of peas, which were different from the original MAR sequences. In order toinvestigate the three MARs on gene expression in transgenic plants, the plant expressionvectors were constructed with udiA gene codingβ-glucuronidase (GUS) which was flankedon both sides by the MARs in the same direction. Quantitative detection of GUS activityshowed that the MARs could increase GUS expression levels in vivo in contrast to thecontrol. The average GUS activity was 4.16 folds for MMAR, 3.66 folds for DMAR and2.08 folds for HMAR, respectively. But the expression differences among the individualtransformants were still obvious.
3.The cotyledons with petiole as acceptors, Chinese cabbage genetic transformationsystem mediated by Agrobacterium tumefaciens was set up and Amaranthus viridis L.agglutinin (AVA) transformed plants that were resistant to Myzus persicae were acquired. Effects of MAR (matrix attachment region) on AVA gene expression in transgentic Chinesecabbage were studied with two different plant expression vectors. The number oftransgenic plants was 29.63% higher than that of the control. The bioassay of Myzuspersicae revealed that transgetic plants showed more or less enhanced resistance andinhibition to population, and the average inhibition rate was 55.8%. Among the transgeticplants that AVA gene was mediated by MAR, the expression level of AVA gene was higherthan that of the control, and the expression variance of AVA gene among the transgeticplants was smaller.
4. According to the insect-resistant characteristics of CpTI and AVAc genes, the plantexpression vector pC2MAR-AVAc/CpTI was constructed. With the embryogenic calli ofsugarcane variety ROC25 as the acceptors, a genetic transformation system mediated byAgrobacterium tumefaciens was established and the fusion insect-resistant genetransformed plants were acquired. The plant expression vector was constructed with thefused gene which was flanked on both sides of the MARs. The plant vectors weretransformed into the embryogenic calli with the plant expression vector without MAR ascontrol. In the genetic transformation of sugarcane, the vector ligated with MARs in thesame direction on both sides of the fused gene could increase the number of transgenic lines,which was 62.5% higher than that of the control. Moreover, the number of transgenicplants regenerated from each resistant callus increased by 34.32%.
引文
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