Cloning And Transgenic Studies On Amaranthus Viridis Agglutinin Gene

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%.