TAT-Cloan-DH 融合肽生物活性分析及在杨树中的表达

Diapause intervenes in all the major stages of the insect life cycle. For most insect species, diapause is a developmental option and individuals have the capacity to determine whether or not they enter that state by monitoring changes in environmental and internal conditions through the mediation of endocrinological mechanisms. Diapause hormone (DH) is a neuropeptide hormone. DH could suggest potential tools for the development of a future pest resistance plant breeding strategy. Nevertheless, it still remains a challenge to deliver the protein to desired targets. However, discovery of the TAT protein transduction domain (PTD) has opened novel avenues for directing in vitro and in vivo delivery of proteins into cells. .Synthetic TAT protein has the unique feature of entering the cultured cell efficiently in vitro with other protein expression of fusion proteins (TAT-protein), so construct fusion genes of DH neuropeptide gene and TAT protein gene and transfrom it to model plants to check its expression and to definitude the synergism of TAT to DH neuropeptide gene in plant system, then break a new using path of TAT and a new way of pests control and plant breeding．In the present study, the cDNA encoding diapause hormone gene from Clostera anastomosis was cloned by RT-PCR. In order to research the relations between the gene and the effect of TAT peptide, the subject would constructed TAT-Cloan-DH-GFP and Cloan-DH-GFP two fusion protein plant expression vectors. First, prokaryotic expression of TAT-Cloan-DH fusion protein will be analysised about the target insect resistance and virulence. Second, agrobacterium mediated transformation to model tree poplar, and the establishment of a complete transformation of the model plant system has made base on further expression．If the studies further verified TAT in plants can improve efficiency and magical effect of pests, and TAT application are of great-theoretical and practical significance, and will open a new way to plant breeding resistance to pests and a new field in neuropeptide hormone application．

滞育有利于昆虫度过不良环境，使群体发育整齐。滞育激素(DH)属昆虫神经肽类分子，其调控滞育行为。通过施加外源DH，使体内激素水平失调，打破昆虫内分泌系统平衡，导致生理机能障碍以至中断正常的活动，进而扰乱害虫群体发育整齐度，可以达到控制害虫的目的。基于已被克隆的分月扇舟蛾滞育激素基因(Cloan-DH)，本研究拟通过构建TAT-Cloan-DH融合基因，原核表达融合肽，借助TAT蛋白转导结构与其它大分子组成融合蛋白，能协助大分子进行跨膜运输的生物功能，分析害虫取食融合蛋白后DH进入虫体的生物过程，通过研究靶标昆虫取食的生理变化机制，阐明TAT与Cloan-DH融合表达的生物活性机理；并将融合基因转化模式树木杨树，分析融合肽在杨树细胞中表达强度与杀虫活性，完成以DH为例的神经肽分子用于抗虫植物培育的方法探索，为植物抗虫分子育种提供理论依据。

在大肠杆菌Escherichia coli BL21（DE3）中表达的TAT-Cloan-DH-EGFP融合蛋白，主要以包涵体形式存在，融合蛋白约占细胞总蛋白含量的18.1%。人工合成TAT-Cloan-DH中部的23个氨基酸多肽免疫家兔制备的抗血清，可用于检测原核表达的融合蛋白TAT-Cloan-DH。将表达TAT-Cloan-DH-EGFP融合蛋白的大肠杆菌E. coli BL21菌体添加到人工饲料中，经供试昆虫幼虫取食后，将不同取食时间（0 h，4 h，8 h，24 h，48 h，72 h和90 h）的试虫制作石蜡切片，结果表明融合蛋白TAT-Cloan-DH-EGFP可以在TAT蛋白转导域的介导下穿过消化道横向跨膜运输，到达虫体其它组织。.通过将TAT-Cloan-DH蛋白注射入供试昆虫的滞育蛹，证实该蛋白具有解除滞育的活性。将人工饲料和分别添加Cloan-DH和TAT-Cloan-DH蛋白的人工饲料饲喂供试昆虫幼虫，结果显示无论在27℃、光周期14L/10D诱导非滞育条件下，还是在20℃、光周期10L/14D诱导滞育条件下，取食融合蛋白TAT-Cloan-DH的供试昆虫幼虫发育历期显著增长，化蛹率显著减少。试验结果进一步证实了TAT蛋白转导域可以介导Cloan-DH进行跨膜转运，并且融合蛋白TAT-Cloan-DH对供试昆虫的生长发育起着抑制作用。.将构建的植物表达载体pBin438-TAT-Cloan-DH，通过农杆菌介导法转化烟草，Western blot 检测表明融合蛋白TAT-Cloan-DH在转基因烟草中获得了表达。在27℃、光周期14L/10D诱导非滞育条件下，试虫取食表达TAT-Cloan-DH融合蛋白的转基因烟草后，幼虫发育历期显著延长，化蛹率也显著减少。结果表明，利用转基因技术将TAT-Cloan-DH基因转化烟草，借助植物表达系统，可以获得具有生物活性的融合蛋白TAT-Cloan-DH，并且转基因烟草使供试昆虫幼虫生长发育减缓，对试虫的生长发育不利。