抗天牛基因（cry3Aa同源基因）的克隆、改造及其对杨树的转化
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摘要
本文通过对中国林业科学研究院林业研究所生物技术室所分离、筛选的苏云金芽孢杆菌(Bacillus thuringiensis)菌株Bt886进行的深入研究，初步确认了该菌株所属的类型，然后深入到分子水平做出了验证。以该菌株为材料，克隆出了对光肩星天牛(Anoplophra labripennis (Motsch.))具有毒杀作用的cry3Aa同源基因，并且对该基因进行了人工改造、人工合成、大肠杆菌表达、生物活性测定、双元表达载体的构建以及对杨树的转化等一系列研究，主要结果如下：
(1)显微观察该菌株所形成的伴孢晶体为方形。生物活性测定结果表明该菌株对榆蓝叶甲(Pyrrhalta aenescens (Fairmaire))和光肩星天牛等鞘翅目昆虫具有较高的毒力，因此初步确认该菌株属于cry3类；
(2)发现该菌株中编码毒蛋白的基因位于质粒上，并且已经成功地克隆到该基因。经测序表明，该cry3Aa同源基因全长1956bp，GC含量较低，为35.95％，与cry3Aa1的序列同源性极高，仅有6个碱基和2个氨基酸的差异；
(3)苏云金芽孢杆菌来源的cry3Aa同源基因与真核来源的杨树基因相比，发现它们对各种氨基酸的同义三联体密码子的偏好和使用频率存住着很大的差异，这可能正是原核来源的基因在真核生物中表达效率低的主要原因之一。通过对氨基酸密码子的偏好、使用频率、多聚腺苷酸信号序列(PPSS)、mRNA不稳定序列(DST)以及Kozak等结构的优化改造，人工设计、合成了预期能在杨树中高效表达的毒蛋白基因Modified cry3Aa同源基因；
(4)利用强启动子表达载体pET30-b完成了cry3Aa同源基因在大肠杆菌中的表达。SDS-PAGE电泳表明，在大肠杆菌中所表达的融合蛋白位于66kDa～97kDa之间；
(5)大肠杆菌表达产物生物测定表明：苏云金芽孢杆菌菌株Bt886对鞘翅目昆虫的高毒力主要归因于菌体内位于质粒上的cry3Aa同源基因。因此，所克隆的cry3Aa同源基因完全可以用于培育抗天牛的植物新品种；
(6)获得了重组双元植物表达载体pBin438-Mcry3和pBI121-2k，为进一步转化杨树及其它植物品种，培育抗天牛的植物新品种奠定了良好的基础；
中南林学院硕士研究生毕业论文2003
（7）建立了两套完整的杨树转化体系，一套为农杆菌介导的外源基因转化体系，一
套为花粉管通道法转化体系；
（）获得了一批抗恃那霉素的，导入了外源基因 err3Aa同源基因和 Modified err3Aa
同源基因的杨树幼茁，目前正在进行检测筛选。
This thesis studied on Bacillus thuringiensis strain Bt886 which was separated and selected by researchers of our laboratory. According to the observation of crystal shape and the bioassay of Motschulsky and Fairmaire, Bacillus thuringiensis strain Bt886 was identified as cry3 type, and the conclusion was assured by the further study on molecular level.
The 1956 base pairs full lengrh homological cry3Aa gene which was toxic to Motschulsky was cloned and sequenced. It is highly homologized to cry3Aal gene. Further sequence analysis show that only 6 base pairs of nucleotide and 2 amino acids are different between them.
The homological cry3Aa gene was expressed in Escherichia coli.. And the expressed products which contain a fused peptide of 66-97 kilo-Dalton was observed by means of SDS-PAGE. Bioassay results show that the expressed protein in Escherichia coli. is toxic to Tenebrio molitor L..
The homological cry3Aa gene was modified according to the characters of poplus genes and the other features of eukaryotic gene, and was artificially synthesized. The original and modified homological c/y3Aa gene were cloned into binary vector pB121 and pBin438 respectively, and transplanted into 84K clone (P. tomentos X P. glandulossa) to breed new anti-insects tree species.
(1)显微观察该菌株所形成的伴孢晶体为方形。生物活性测定结果表明该菌株对榆蓝叶甲(Pyrrhalta aenescens (Fairmaire))和光肩星天牛等鞘翅目昆虫具有较高的毒力，因此初步确认该菌株属于cry3类；
(2)发现该菌株中编码毒蛋白的基因位于质粒上，并且已经成功地克隆到该基因。经测序表明，该cry3Aa同源基因全长1956bp，GC含量较低，为35.95％，与cry3Aa1的序列同源性极高，仅有6个碱基和2个氨基酸的差异；
(3)苏云金芽孢杆菌来源的cry3Aa同源基因与真核来源的杨树基因相比，发现它们对各种氨基酸的同义三联体密码子的偏好和使用频率存住着很大的差异，这可能正是原核来源的基因在真核生物中表达效率低的主要原因之一。通过对氨基酸密码子的偏好、使用频率、多聚腺苷酸信号序列(PPSS)、mRNA不稳定序列(DST)以及Kozak等结构的优化改造，人工设计、合成了预期能在杨树中高效表达的毒蛋白基因Modified cry3Aa同源基因；
(4)利用强启动子表达载体pET30-b完成了cry3Aa同源基因在大肠杆菌中的表达。SDS-PAGE电泳表明，在大肠杆菌中所表达的融合蛋白位于66kDa～97kDa之间；
(5)大肠杆菌表达产物生物测定表明：苏云金芽孢杆菌菌株Bt886对鞘翅目昆虫的高毒力主要归因于菌体内位于质粒上的cry3Aa同源基因。因此，所克隆的cry3Aa同源基因完全可以用于培育抗天牛的植物新品种；
(6)获得了重组双元植物表达载体pBin438-Mcry3和pBI121-2k，为进一步转化杨树及其它植物品种，培育抗天牛的植物新品种奠定了良好的基础；
中南林学院硕士研究生毕业论文2003
（7）建立了两套完整的杨树转化体系，一套为农杆菌介导的外源基因转化体系，一
套为花粉管通道法转化体系；
（）获得了一批抗恃那霉素的，导入了外源基因 err3Aa同源基因和 Modified err3Aa
同源基因的杨树幼茁，目前正在进行检测筛选。
This thesis studied on Bacillus thuringiensis strain Bt886 which was separated and selected by researchers of our laboratory. According to the observation of crystal shape and the bioassay of Motschulsky and Fairmaire, Bacillus thuringiensis strain Bt886 was identified as cry3 type, and the conclusion was assured by the further study on molecular level.
The 1956 base pairs full lengrh homological cry3Aa gene which was toxic to Motschulsky was cloned and sequenced. It is highly homologized to cry3Aal gene. Further sequence analysis show that only 6 base pairs of nucleotide and 2 amino acids are different between them.
The homological cry3Aa gene was expressed in Escherichia coli.. And the expressed products which contain a fused peptide of 66-97 kilo-Dalton was observed by means of SDS-PAGE. Bioassay results show that the expressed protein in Escherichia coli. is toxic to Tenebrio molitor L..
The homological cry3Aa gene was modified according to the characters of poplus genes and the other features of eukaryotic gene, and was artificially synthesized. The original and modified homological c/y3Aa gene were cloned into binary vector pB121 and pBin438 respectively, and transplanted into 84K clone (P. tomentos X P. glandulossa) to breed new anti-insects tree species.
引文
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