摘要
甘蔗(Saccharum officinarum L.)是重要的糖料和能源作物,甘蔗生产中螟虫(鳞翅目)、甘蔗绵蚜(同翅目)以及蛴螬(鞘翅目)等害虫的危害已成为其持续、稳定和健康发展的严重障碍。为了使甘蔗抗虫转基因的研究工作更有针对性,我们对海南甘蔗虫害的种类及其危害做了全面的调查,其中红尾白螟、二点螟、条螟、粉蚧、绵蚜,蔗根象等危害最严重,而这些害虫主要集中于鳞翅目、同翅目两大类，该调查结果与全国甘蔗蔗区害虫的危害情况基本一致。
在甘蔗抗虫基因工程中,通过将抗虫蛋白基因导入甘蔗来提高甘蔗对虫害的抵抗能力已成为一种直接而有效的方法。随着越来越多抗虫蛋白被发现,人们都试图采用多基因共表达的策略将多种抗虫蛋白基因转入植物,以提高植物的抗虫能力和扩大抗虫谱。但是,山于受到可选择的载体数目和载体容量的限制,该目的很难较好的实现。融合基因技术的发展成为解决这一难题的有效途径。通过构建融合基因,可以实现多个基因的同步、同水平表达。正是这些优点,融合基因技术越来越受到人们的关注。针对甘蔗生产中鳞翅目、同翅目两大类主要害虫,本研究选择Bt CryⅠA (c)和gna两个已经证明具有很强抗螟虫和蚜虫能力的抗虫蛋白,采用重叠延伸PCR技术(gene splicing by overlap extension PCR,简称SOE PCR),通过连接多肽FMDV2A序列将CryⅠA(c)和gna基因相融合,得到CryⅠA (c)-2A-gna抗虫融合基因,通过农杆菌介导转化甘蔗,实现两个蛋白在甘蔗体内的共表达,以求达到提高甘蔗对虫害的抵抗能力的目的。主要实验结果如下:
(1)对海南甘蔗产区5个县(市)16个乡镇进行害虫调查,初步摸清了海南甘蔗害虫种类,共发现害虫9日35科58种,其中鳞翅目10科15种,半翅目4科5种，同翅目7科9种,鞘翅目7科12种,直翅目4科12种,蜚蠊目、等翅目、缨翅目和柄眼目各1科1种。分析了甘蔗生产的主要害虫分布、危害特点、发生规律。其中红尾白螟、二点螟、条螟、粉蚧、绵蚜,蔗根象等危害最严重；
(2)将CryⅠA (c)和gna基因用一段具有自我剪切功能的FMDV2A序列连接子连接,成功了融合CryⅠA (c)-2A-gna基因；
(3)构建了含有融合基因CryⅠA (c)-2A-gna的植物表达载体pNUBG.同时构建了CryⅠA (c)和CrylA (b)两个单独基因的植物表达载体pUBTC和pUBTB,获得的三个植物表达载体用“冻融法”将其导入根癌农杆菌EHA105中；
(4)将三个表达载体分别通过农杆菌介导法对甘蔗进行遗传转化：获得转融合基因CryⅠA (c)-2A-gna抗性植株196株,转CryⅠA (c)基因86株,转CryⅠA (b)基因抗性植株78株。所得的抗性植株进行PCR检测,转C ryⅠA (c)-2A-gna融合基因抗性植株呈阳性的有135株,转CryⅠA (c)基因的57株,转CryⅠA(b)基因有52株,初步证明外源基因已整合到甘蔗的基因组中；
(5)选取转融合基因CryⅠA (c)-2A-gna的6株PCR呈阳性的植株进行Southern blot检测,植株1,2,3,5,6有杂交信号,单拷贝,证明外源基因已整合到甘蔗的基因组中,只有植株4的无杂交信号；
(6)选取转融合基因CryⅠA (c)-2A-gna的5株Southern blot呈阳性的植株进行RT-PCR检测,5株转基因甘蔗的外源基因CryⅠA (c)-2A-gna基因在转录水平上得到了表达；
(7)选取转融合基因CryⅠA (c)-2A-gna的5株Southern blot检测呈阳性的植株提取植物粗蛋白,进行GNA凝血.实验，说明融合基因CryⅠA (c)-2A-gna中的gna基因在转基因甘蔗植株中有不同程度的表达；
(8)选取转融合基因CryⅠA (c)-2A-gna的5株Southern blot检测呈阳性的植株提取植物粗蛋白,利用Bt-Cry1Ab/Ac免疫检测试纸条进行Bt杀虫蛋白检测,全部表现为阳性结果，有Bt-Cry1Ab/Ac免疫检测试纸条上出现了两条紫红色条带,一条质控线,一条检测线,说明融合基因CryⅠA (c)-2A-gna中的CryⅠA基因在转基因甘蔗植株中得到表达。
Sugarcane (Saccharum officinarum L.) is an important cash crop on producing sugar and energy. Pests, such as Sugarcane borer (Lepidopter)、Sugarcane cottony aphid (Homoptera)and Grubs (Coleoptera), have severely restricted the sustainable and stable development of sugarcane industry. For better doing research on anti-pests transgenic sugarcane, we investigated the pests kinds and hazard state occurring on sugarcane in Hainan province, the most harmful insects including Tryporyza intacta Snellen, Hieroglyphus tonkinensis I. Bolivar and Episomoides albinus Matsumura, belong to Lepidopter、Homoptera. Sugarcane pests occurring in Hainan province were identical with nationwide other regions planting sugarcane.
With the development of insect-resistant gene engineering, transferring anti-pests genes into sugarcane genome has been a direct and effective pathway to pests'control. For more and more anti-pests proteins discovered, multi-tandem anti-pests genes could be transferred to plants for improving the insect resistance and expanding insect resistant spectrum.
However, it is difficult to transfer multi-gene simultaneously because of the limitation on vectors number and capacity. Gene fusion is an efficient way to solve this problem. Constructing fusion genes can make multi-genes expressed simultaneously on the same level. Just because of these advantages, gene fusion technology has been applied widely. Towards the two main pests species Lepidopter、Homoptera on sugarcane, BtCrylA (c) and gna proved of great resistance were selected to construct fusion genes. BtCryⅠA (c) and gna were linked by connecting peptide FMDV2A forming CryⅠA (c)-2A-gna gene using gene splicing by overlap extension PCR. As a result, BtCrylA (c) and GNA can express together in sugarcane to increase sugarcane resistance to borer and aphid. The main results are as follows:
(1) After investigation on sugarcane pests in 16 towns in 5 counties of Hainan Province, we basically clarified the pests'species occurring on sugarcane. A total of 58 pests species were identified belonging to 36 families in 9 orders:15 species in 10 families of Lepidoptera order,5 species in 4 families of Hemiptera order,9 species in 7 families of Homoptera order, 12 species in 11 families of Coleoptera order, One Blattodea species,1 Isoptera species,1 Thysanoptera species and 1 mollusk. Tryporyza intacta Snellen, Hieroglyphus tonkinensis I. Bolivar and Episomoides albinus Matsumura were the harmful sugarcane pests in Hainan but few reports in other provinces in sugarcane. The pests distribution, hazard Characterization and occurrence regularity were analyzed.
(2) BtCryⅠA (c) and gna was connected by a self-slice sequence FMDV2A linker, constructed fusion gene CryⅠA (c)-2A-gna.
(3) The plant expression vector containing the fusion gene was constructed,and named pNUBG respectively.In addition,another two vectors containing CryⅠA (c) and CryⅠA (b),respectively,were constructed as control.Then these expression vectors were introduced into Agrobacterium tumefaciens EHA105 by freezing-melting transformation method.
(4) Three expression vectors were introduced into sugarcane by Agrobatorium-mediated transformation, and 135 PCR positive plants containing CryⅠA (c)-2A-gna constructs,57 PCR positive plants containing CryⅠA (c) and 52 PCR positive plants containing CryⅠA (b) were obtained. These results showed that there foreign genes had been probably integrated into sugarcane genome.
(5) Six CryⅠA (c)-2A-gna PCR positive plants were selected to detect by Southern blot, The resoults of Southern blot were five transgenic lines all showed hybridization bands,One line showed no hybridization band.
(6) Five CryⅠA (c)-2A-gna Southern blot positive plants were detected by RT-PCR, and each plant expressed CryⅠA (c)-2A-gna on transcription level.
(7) Extracted raw proteins used to red blood cell agglutination bioassay from five Southern blot positive plants and, the results indicated that GNA were expressed in part transferred plants at different degree.
(8) Extracted raw proteins from five Southern blot positive plants to detect Bt insecticidal protein by Bt immune detection strips and, the results indicated that all plants showed positive.
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