双Bt基因对杨树的遗传转化及外源基因表达研究
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摘要
为鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫同时具有较强抗性的株系，明确联合使用两种或两种以上的抗虫基因的抗虫效果。以8个转三基因双Bt （Cry3Aa+Cry1Ac+API）741杨株系、1个转双抗虫基因（Cry1Ac+API）741杨株系和3个转单抗虫基因（Cry3Aa）741杨株系为实验材料，未转基因741杨为对照，进行了外源基因表达测定和抗虫性对比试验。同时借助农杆菌介导，采用共转化法，用构建在同一表达载体pCAMBIA1305-Cry1Ac-Cry3Aa上的双Bt基因对巨霸杨进行遗传转化，获得了4株潮霉素抗性植株。经过PCR初步检测，表明双Bt基因已经整合进了巨霸杨基因组中。主要研究结果如下：
1.转抗虫基因741杨分别为：单转Bt Cry3Aa基因741杨pCC系列3个株系pCC11、pCC53、pCC84；转双抗虫基因（Cry1Ac+API）741杨1个株系pB29；在pB29基础上通过二次转化法将Cry3Aa基因转入获得的转三基因双Bt（Cry3Aa+Cry1Ac+API）741杨pCCA系列8个株系pCCA1、pCCA2、pCCA3、pCCA4、pCCA5、pCCA6、pCCA7和pCCA9。对各转基因株系及对照进行组培快繁，筛选确立了诱导741杨分化和生根的适宜培养基。分化培养基为：MS+6BA1.0mg·L~(-1)+NAA0.1mg·L~(-1)；生根培养基为：1/2MS+IBA0.3mg·L~(-1)。
2.转双Bt基因741杨等13个参试材料经特异引物PCR扩增Cry1Ac基因和Cry3Aa基因： pB29和转双Bt基因pCCA系列均扩增得到了Cry1Ac基因特异条带，对照741和pCC系列基因组未出现扩增条带。pCC系列和转双Bt基因pCCA系列均得到了Cry3Aa基因特异条带，对照741和pB29基因组未出现扩增条带。说明pB29中Cry1Ac基因和pCC系列中的Cry3Aa基因稳定存在。在pB29基础上，通过二次介导法外源Cry3Aa基因已整合到pCCA各无性系基因组中。
3.通过RT-PCR和荧光定量PCR结合，在转录水平对外源基因的表达进行了检测。实时荧光监测表明：8个双Bt株系即检测到了Cry1Ac荧光扩增信号又检测到了Cry3Aa基因的荧光扩增信号。pCC~(-1)1、53、84只有Cry3Aa基因的荧光信号表达，而pB29只有Cry1Ac基因的荧光信号表达，对照741没有显现双Bt基因的荧光扩增信号。根据测得的Ct值，计算出了各样品中Cry1Ac基因和Cry3Aa基因在mRNA转录本中的表达量。双Bt株系pCCA系列及pB29中Cry1Ac基因的起始表达量在3.26×104~7.50×105之间；双Bt株系pCCA系列及pCC系列Cry3Aa基因的起始表达量在1.79×108~6.05×109之间。数据显示Cry3Aa基因的起始表达量在108~109数量级，比Cry1Ac的起始表达量（104~105数量级）高出了一万倍。
4.用Bt-Cry1Ab/1Ac和Bt-Cry3A ELISA蛋白检测试剂盒，检测Cry1Ac蛋白：双Bt741杨pCCA系列的8个系号和pB29呈现蓝色阳性反应，蛋白含量在16.44~60.32ng·g~(-1)（FW）；pCC系列和对照741无显色反应。检测Cry3Aa蛋白：双Bt741杨8个系号及pCC系列呈现黄色阳性反应，蛋白含量在2.24~13.30μg·g~(-1)（FW）；pB29和对照741无显色反应。检测结果表明，双Bt株系能同时表达两种Bt毒蛋白，单Bt株系也表达了与各自所含基因相应的蛋白。从毒蛋白表达量看Cry3Aa蛋白表达量远远高于Cry1Ac蛋白表达量。Cry3Aa蛋白表达量在微克级，Cry1Ac蛋白表达量在纳克级。
5.用转基因株系新鲜叶片进行柳蓝叶甲（Plagiodera versicolora）1~3龄幼虫和成虫及美国白蛾（Hyphantria cunea）1龄和4龄幼虫室内饲虫实验表明：转入不同抗虫基因的杨树对昆虫的抗性具有选择性，对非靶标昆虫没有毒杀作用。转双Bt基因741杨对鳞翅目的美国白蛾和鞘翅目的柳蓝叶甲具有双抗性，不同转基因株系表现出高中低的抗性水平。pCCA~(-1)、2、5、6、9对柳蓝叶甲表现高抗，pCCA-3、4、7表现中低抗性。5个高抗株系的抗性水平明显比单Bt Cry3Aa基因的3个高抗株系（pCC~(-1)1、53、84）的抗虫性高，用这5个系号饲喂的柳蓝叶甲成虫3天时60%以上死亡，5天时死亡率达到85%~100%；而pCC的3个系号3天时死亡率在50%以下，5天时也只有60%~70%。在对美国白蛾的抗性上，有7个双Bt株系（pCCA2-7和pCCA9）的抗性水平与pB29表现一致，4龄幼虫在第7~9天时死亡率达100%；只有1个系号pCCA1对美国白蛾表现出了极低的抗性，4龄幼虫在第7天和第9天时的死亡率分别只有7%和23%。pCC系列不含抗鳞翅目的Bt Cry1Ac基因，对美国白蛾未表现抗虫性。
6.饲虫结果还表明无论是柳蓝叶甲还是美国白蛾，1龄幼虫对Bt毒蛋白的耐受性比较差，取食2~3天全部死亡；柳蓝叶甲成虫及美国白蛾4龄幼虫耐受性明显增强，取食可延长到7~11天。通过对取食叶片实时拍照记录的取食危害面积来看，转基因株系同对照比，即使中低抗株系也能对叶片起到很好的保护作用。
7.对美国白蛾4龄幼虫的总排粪量和体长调查表明：低抗株系pCCA1的总排粪量是高抗株系pCCA2-7、pCCA9和pB29的7~23倍，但跟对照的总排粪量比，只有对照的25%。从体长来看，未转基因741杨上的美国白蛾幼虫体长达到了19mm，pCCA2-7、pCCA9和pB29的体长在10~12mm之间，而取食低抗株系pCCA1的最后体长也只有13mm（为对照的68%）。从虫粪和体长这两个指标分析来看，充分说明了转基因植株对昆虫的生长发育起到了明显的抑制作用。
8.采用农杆菌介导法，以潮霉素做筛选标记，用构建在同一表达载体上的双Bt基因（Cry3Aa+Cry1Ac）对巨霸杨进行遗传转化。实验对诱导叶片分化适宜培养基和适宜潮霉素浓度这两个影响转化的关键因素进行了深入研究。确定诱导叶片分化不定芽的最佳培养基为MS+6BA0.25mg·L~(-1)+IBA0.1mg·L~(-1)。通过潮霉素在0mg·L~(-1)~15mg·L~(-1)范围内不同浓度梯度对叶片分化和茎尖生长影响的实验，确定诱导抗性芽分化的潮霉素临界筛选浓度为5mg·L~(-1)。经过对再生抗性芽的多次潮霉素筛选，获得抗性稳定的转双Bt基因巨霸杨无性系4个，编号为JB1、JB2、JB3和JB4。
9.用特异引物分别对获得的转双Bt基因巨霸杨无性系进行PCR检测，结果显示：JB1、JB2、JB3和JB4均扩增得到一条与阳性质粒作模板PCR扩增条带大小相同的749bp（Cry1Ac基因）和612bp（Cry3Aa基因）的特异条带，而对照未转化巨霸杨基因组未出现PCR扩增特异条带。初步证明双Bt基因已经整合进了巨霸杨基因组中。
With the aim of identifying and selecting double Bt transgenic741poplar lines with high resistance against to both Coleoptera and Lepidopterainsects, studying the effects of combining two or more insect-resistance genesin plants, eight741poplar lines with three insect resistance genes(Cry3Aa+Cry1Ac+API), one741poplar with two insect resistance genes(Cry1Ac+API), three741poplar lines with one insect resistance genes(Cry3Aa) were selected as materials and non-transgenic741poplar as control.Comparative studies were conducted on exogenous gene expression and theassessment of insect resistant ability. At the same time, double Bt genes invector pCAMBIA1305-Cry1Ac-Cry3Aa were genetically transformed intoJuBa poplar using Agrobacterium mediated co-transformation method. Fourlines showed hygromycin resistance. PCR detection preliminary proved that double Bt sequneces have successfully recombined into the genome of JuBapoplar. The main results are as follows:
1. The insect-resistance transgenic741poplars were the following series:pCC series with single Bt gene (Cry3Aa): pCC11, pCC53and pCC84; pB29with double insect resistance genes (Cry1Ac+API); pCCA series with threeinsect resistance genes (Cry3Aa+Cry1Ac+API): pCCA1, pCCA2, pCCA3,pCCA4, pCCA5, pCCA6, pCCA7, pCCA9. All transgenic lines and thecontrol were propagated through tissue culture. The suitable differentiation(MS+6BA1.0mg·L~(-1)+NAA0.1mg·L~(-1)) and rooting (1/2MS+IBA0.3mg·L~(-1))medium were selected and established.
2. The presence of the Cry1Ac gene and the Cry3Aa gene in these741plants were detected by PCR analyses with special primers. The target bandsof Cry1Ac gene in pB29and pCCA series appeared as the plasmid positivetemplate. No target bands showed in the control and pCC series. The targetbands of Cry3Aa gene in pCC series and pCCA series appeared as the plasmidpositive template. No target bands were detected in the control and pB29genome. The results indicated that Cry1Ac gene stably existed in pB29andCry3Aa gene stably existed in pCC series. Cry3Aa gene had been integratedinto the genome of pCCA series on base of pB29.
3. The exogenous gene expression was analyzed at the transcription levelby the combination of RT-PCR and real-time quantitative PCR. Real-timePCR showed that both Cry1Ac gene and Cry3Aa gene presented in theamplification fluorescence signal of eight double Bt lines. pCC~(-1)1,53,84only had Cry3Aa gene amplification signal and pB29only had Cry1Ac genesignal expression. No any amplification signal was detected in control741.The primary transcript of Bt genes in mRNA were calculated according to themeasured Ct value. The primary transcript of Cry1Ac gene of pB29andpCCA series were ranged from3.26×10~4to7.50×10~5. The primary transcriptof Cry3Aa gene of pCC series and pCCA series were ranged from1.79×10~8to6.05×10~9. The results showed that the primary transcript of Cry3Aa gene(108~109order of magnitude) was ten thousand times higher than that ofCry1Ac gene (104~105order of magnitude).
4. Bt-Cry1Ab/1Ac and Bt-Cry3A ELISA kit were used to detected theexpressions of insecticidal proteins in each transgenic lines. pB29and eightdouble Bt pCCA series showed blue positive reaction of Cry1Ac protein. Theprotein contents ranged from16.44ng·g~(-1)(FW) to60.32ng·g~(-1)(FW). pCC~(-1)1,53,84and the CK741showed no color reaction. As to Cry3Aa protein, eightdouble Bt pCCA series and pCC~(-1)1,53,84showed yellow positive reaction. The protein contents ranged from2.24μg·g~(-1)(FW) to13.30μg·g~(-1)(FW). pB29and the CK741showed no color reaction. Test results indicated that doubleBt lines could express double Bt protein (Cry1Ac and Cry3Aa protein), singleBt lines also expressed corresponding proteins. The content of Cry3Aa(microgram order of magnitude) was much higher than that of Cry1Ac protein(nanogram order of magnitude).
5. Toxicity evaluation tests were performed in the laboratory withPlagiodera versicolora (L1~L3larvae and adult) and Hyphantria cunea (L1and L4larvae) on fresh detached leaves. Transgenic741poplar lines carryingdifferent insect-resistance genes demonstrated selective resistance to targetinsects, but showed no toxic effects towards non-target insects.741lines withdouble Bt transgenes had double insect-resistance ability, and individual linesshowed resistance ranging from high, medium to low. pCCA~(-1)、2、5、6、9showed high resistance to P. versicolora and pCCA-3、4、7showed mediumresistance. The five high resistance lines showed higher toxicity than threesingle Cry3Aa gene lines with high resistance (pCC~(-1)1,53,84). The mortalityof adults feeded on these five lines was above60%at the third day andreached85%~100%at the fifth day, while the mortality feeded on three pCCseries was below50%at the third day and only60%~70%at the fifth day. In regards of H. cunea, seven lines (pCCA2pCCA7and pCCA9) exhibitedsimilar effectiveness as the single Bt line (pB29). The mortality of L4larvaereached100%at the7th~9th day. Only one line (pCCA1) showed anextremely low level of resistance. The mortality of L4larvae was7%and23%at the seventh day and the nineth day separately. pCC series showed notoxicity to H. cunea without Bt cry1Aa gene.
6. Feeding tests also showed either P. versicolora or H. cunea, L1larvaehad low tolerance to Bt toxic protein and died within2~3days. As for adult P.versicolora and L4larvae of H. cunea, tolerance increased obviously byextending the feed time to7~11days. From the demaged leaf area recordedby photos we can see that, even transgenic lines with midium and lowresistance level, all could perform a good protection of their leaves comparingto the control741plant.
7. The investigation of total frass and body lenghth of L4H. cunea larvaeshowed that the total frass of pCCA1was7~23times the weight of highinsect-resistance lines. Compared with that of the control741, only occupied25%of the contrast. About the body length, H. cunea larva feeded bynon-transgenic741poplar reached19mm; body length feeded by pCCA2-7,pCCA9and pB29was10~12mm, and that of low insect-resistance pCCA1 was only13mm. From the analysis of frass and body length illustrated thatthe transgenic lines had inhibited of growth and development of the larvea.
8. By Agrobacterium-mediated method and hygromycin (Hyg) asselectable marker, the genetic transformation of JuBa poplar with double Bt(Cry3Aa+Cry1Ac) genes was studied. The suitable differentiation mediumand the suitable Hyg concentration were deeply investigated as two keyfactors influencing the transformation efficiency. The best differentiationmedium was established as: MS+6BA0.25mg·L~(-1)+IBA0.1mg·L~(-1). The effectof different Hyg concentration (0mg·L~(-1)~15mg·L~(-1)) on leaves and shootswere investigated. Hyg5mg·L~(-1)was determined for the critical concentration.Through several successive transfer and selection in the medium containingHyg,4lines with stable Hyg resistance were obtained and named JB1, JB2,JB3and JB4.
9. The presence of the Cry1Ac gene and the Cry3Aa gene in these fourJuBa plants were detected by PCR analyses with special primers. The resultshowed that the target electrophoretic749bp bands of Cry1Ac gene and612bp bands of Cry3Aa gene in JB1, JB2, JB3and JB4appeared as the plasmidpositive template, while there was no target band in the control plant, preliminary indicating that the double Bt genes had been integrated into thegenome of JuBa poplar.
1.转抗虫基因741杨分别为：单转Bt Cry3Aa基因741杨pCC系列3个株系pCC11、pCC53、pCC84；转双抗虫基因（Cry1Ac+API）741杨1个株系pB29；在pB29基础上通过二次转化法将Cry3Aa基因转入获得的转三基因双Bt（Cry3Aa+Cry1Ac+API）741杨pCCA系列8个株系pCCA1、pCCA2、pCCA3、pCCA4、pCCA5、pCCA6、pCCA7和pCCA9。对各转基因株系及对照进行组培快繁，筛选确立了诱导741杨分化和生根的适宜培养基。分化培养基为：MS+6BA1.0mg·L~(-1)+NAA0.1mg·L~(-1)；生根培养基为：1/2MS+IBA0.3mg·L~(-1)。
2.转双Bt基因741杨等13个参试材料经特异引物PCR扩增Cry1Ac基因和Cry3Aa基因： pB29和转双Bt基因pCCA系列均扩增得到了Cry1Ac基因特异条带，对照741和pCC系列基因组未出现扩增条带。pCC系列和转双Bt基因pCCA系列均得到了Cry3Aa基因特异条带，对照741和pB29基因组未出现扩增条带。说明pB29中Cry1Ac基因和pCC系列中的Cry3Aa基因稳定存在。在pB29基础上，通过二次介导法外源Cry3Aa基因已整合到pCCA各无性系基因组中。
3.通过RT-PCR和荧光定量PCR结合，在转录水平对外源基因的表达进行了检测。实时荧光监测表明：8个双Bt株系即检测到了Cry1Ac荧光扩增信号又检测到了Cry3Aa基因的荧光扩增信号。pCC~(-1)1、53、84只有Cry3Aa基因的荧光信号表达，而pB29只有Cry1Ac基因的荧光信号表达，对照741没有显现双Bt基因的荧光扩增信号。根据测得的Ct值，计算出了各样品中Cry1Ac基因和Cry3Aa基因在mRNA转录本中的表达量。双Bt株系pCCA系列及pB29中Cry1Ac基因的起始表达量在3.26×104~7.50×105之间；双Bt株系pCCA系列及pCC系列Cry3Aa基因的起始表达量在1.79×108~6.05×109之间。数据显示Cry3Aa基因的起始表达量在108~109数量级，比Cry1Ac的起始表达量（104~105数量级）高出了一万倍。
4.用Bt-Cry1Ab/1Ac和Bt-Cry3A ELISA蛋白检测试剂盒，检测Cry1Ac蛋白：双Bt741杨pCCA系列的8个系号和pB29呈现蓝色阳性反应，蛋白含量在16.44~60.32ng·g~(-1)（FW）；pCC系列和对照741无显色反应。检测Cry3Aa蛋白：双Bt741杨8个系号及pCC系列呈现黄色阳性反应，蛋白含量在2.24~13.30μg·g~(-1)（FW）；pB29和对照741无显色反应。检测结果表明，双Bt株系能同时表达两种Bt毒蛋白，单Bt株系也表达了与各自所含基因相应的蛋白。从毒蛋白表达量看Cry3Aa蛋白表达量远远高于Cry1Ac蛋白表达量。Cry3Aa蛋白表达量在微克级，Cry1Ac蛋白表达量在纳克级。
5.用转基因株系新鲜叶片进行柳蓝叶甲（Plagiodera versicolora）1~3龄幼虫和成虫及美国白蛾（Hyphantria cunea）1龄和4龄幼虫室内饲虫实验表明：转入不同抗虫基因的杨树对昆虫的抗性具有选择性，对非靶标昆虫没有毒杀作用。转双Bt基因741杨对鳞翅目的美国白蛾和鞘翅目的柳蓝叶甲具有双抗性，不同转基因株系表现出高中低的抗性水平。pCCA~(-1)、2、5、6、9对柳蓝叶甲表现高抗，pCCA-3、4、7表现中低抗性。5个高抗株系的抗性水平明显比单Bt Cry3Aa基因的3个高抗株系（pCC~(-1)1、53、84）的抗虫性高，用这5个系号饲喂的柳蓝叶甲成虫3天时60%以上死亡，5天时死亡率达到85%~100%；而pCC的3个系号3天时死亡率在50%以下，5天时也只有60%~70%。在对美国白蛾的抗性上，有7个双Bt株系（pCCA2-7和pCCA9）的抗性水平与pB29表现一致，4龄幼虫在第7~9天时死亡率达100%；只有1个系号pCCA1对美国白蛾表现出了极低的抗性，4龄幼虫在第7天和第9天时的死亡率分别只有7%和23%。pCC系列不含抗鳞翅目的Bt Cry1Ac基因，对美国白蛾未表现抗虫性。
6.饲虫结果还表明无论是柳蓝叶甲还是美国白蛾，1龄幼虫对Bt毒蛋白的耐受性比较差，取食2~3天全部死亡；柳蓝叶甲成虫及美国白蛾4龄幼虫耐受性明显增强，取食可延长到7~11天。通过对取食叶片实时拍照记录的取食危害面积来看，转基因株系同对照比，即使中低抗株系也能对叶片起到很好的保护作用。
7.对美国白蛾4龄幼虫的总排粪量和体长调查表明：低抗株系pCCA1的总排粪量是高抗株系pCCA2-7、pCCA9和pB29的7~23倍，但跟对照的总排粪量比，只有对照的25%。从体长来看，未转基因741杨上的美国白蛾幼虫体长达到了19mm，pCCA2-7、pCCA9和pB29的体长在10~12mm之间，而取食低抗株系pCCA1的最后体长也只有13mm（为对照的68%）。从虫粪和体长这两个指标分析来看，充分说明了转基因植株对昆虫的生长发育起到了明显的抑制作用。
8.采用农杆菌介导法，以潮霉素做筛选标记，用构建在同一表达载体上的双Bt基因（Cry3Aa+Cry1Ac）对巨霸杨进行遗传转化。实验对诱导叶片分化适宜培养基和适宜潮霉素浓度这两个影响转化的关键因素进行了深入研究。确定诱导叶片分化不定芽的最佳培养基为MS+6BA0.25mg·L~(-1)+IBA0.1mg·L~(-1)。通过潮霉素在0mg·L~(-1)~15mg·L~(-1)范围内不同浓度梯度对叶片分化和茎尖生长影响的实验，确定诱导抗性芽分化的潮霉素临界筛选浓度为5mg·L~(-1)。经过对再生抗性芽的多次潮霉素筛选，获得抗性稳定的转双Bt基因巨霸杨无性系4个，编号为JB1、JB2、JB3和JB4。
9.用特异引物分别对获得的转双Bt基因巨霸杨无性系进行PCR检测，结果显示：JB1、JB2、JB3和JB4均扩增得到一条与阳性质粒作模板PCR扩增条带大小相同的749bp（Cry1Ac基因）和612bp（Cry3Aa基因）的特异条带，而对照未转化巨霸杨基因组未出现PCR扩增特异条带。初步证明双Bt基因已经整合进了巨霸杨基因组中。
With the aim of identifying and selecting double Bt transgenic741poplar lines with high resistance against to both Coleoptera and Lepidopterainsects, studying the effects of combining two or more insect-resistance genesin plants, eight741poplar lines with three insect resistance genes(Cry3Aa+Cry1Ac+API), one741poplar with two insect resistance genes(Cry1Ac+API), three741poplar lines with one insect resistance genes(Cry3Aa) were selected as materials and non-transgenic741poplar as control.Comparative studies were conducted on exogenous gene expression and theassessment of insect resistant ability. At the same time, double Bt genes invector pCAMBIA1305-Cry1Ac-Cry3Aa were genetically transformed intoJuBa poplar using Agrobacterium mediated co-transformation method. Fourlines showed hygromycin resistance. PCR detection preliminary proved that double Bt sequneces have successfully recombined into the genome of JuBapoplar. The main results are as follows:
1. The insect-resistance transgenic741poplars were the following series:pCC series with single Bt gene (Cry3Aa): pCC11, pCC53and pCC84; pB29with double insect resistance genes (Cry1Ac+API); pCCA series with threeinsect resistance genes (Cry3Aa+Cry1Ac+API): pCCA1, pCCA2, pCCA3,pCCA4, pCCA5, pCCA6, pCCA7, pCCA9. All transgenic lines and thecontrol were propagated through tissue culture. The suitable differentiation(MS+6BA1.0mg·L~(-1)+NAA0.1mg·L~(-1)) and rooting (1/2MS+IBA0.3mg·L~(-1))medium were selected and established.
2. The presence of the Cry1Ac gene and the Cry3Aa gene in these741plants were detected by PCR analyses with special primers. The target bandsof Cry1Ac gene in pB29and pCCA series appeared as the plasmid positivetemplate. No target bands showed in the control and pCC series. The targetbands of Cry3Aa gene in pCC series and pCCA series appeared as the plasmidpositive template. No target bands were detected in the control and pB29genome. The results indicated that Cry1Ac gene stably existed in pB29andCry3Aa gene stably existed in pCC series. Cry3Aa gene had been integratedinto the genome of pCCA series on base of pB29.
3. The exogenous gene expression was analyzed at the transcription levelby the combination of RT-PCR and real-time quantitative PCR. Real-timePCR showed that both Cry1Ac gene and Cry3Aa gene presented in theamplification fluorescence signal of eight double Bt lines. pCC~(-1)1,53,84only had Cry3Aa gene amplification signal and pB29only had Cry1Ac genesignal expression. No any amplification signal was detected in control741.The primary transcript of Bt genes in mRNA were calculated according to themeasured Ct value. The primary transcript of Cry1Ac gene of pB29andpCCA series were ranged from3.26×10~4to7.50×10~5. The primary transcriptof Cry3Aa gene of pCC series and pCCA series were ranged from1.79×10~8to6.05×10~9. The results showed that the primary transcript of Cry3Aa gene(108~109order of magnitude) was ten thousand times higher than that ofCry1Ac gene (104~105order of magnitude).
4. Bt-Cry1Ab/1Ac and Bt-Cry3A ELISA kit were used to detected theexpressions of insecticidal proteins in each transgenic lines. pB29and eightdouble Bt pCCA series showed blue positive reaction of Cry1Ac protein. Theprotein contents ranged from16.44ng·g~(-1)(FW) to60.32ng·g~(-1)(FW). pCC~(-1)1,53,84and the CK741showed no color reaction. As to Cry3Aa protein, eightdouble Bt pCCA series and pCC~(-1)1,53,84showed yellow positive reaction. The protein contents ranged from2.24μg·g~(-1)(FW) to13.30μg·g~(-1)(FW). pB29and the CK741showed no color reaction. Test results indicated that doubleBt lines could express double Bt protein (Cry1Ac and Cry3Aa protein), singleBt lines also expressed corresponding proteins. The content of Cry3Aa(microgram order of magnitude) was much higher than that of Cry1Ac protein(nanogram order of magnitude).
5. Toxicity evaluation tests were performed in the laboratory withPlagiodera versicolora (L1~L3larvae and adult) and Hyphantria cunea (L1and L4larvae) on fresh detached leaves. Transgenic741poplar lines carryingdifferent insect-resistance genes demonstrated selective resistance to targetinsects, but showed no toxic effects towards non-target insects.741lines withdouble Bt transgenes had double insect-resistance ability, and individual linesshowed resistance ranging from high, medium to low. pCCA~(-1)、2、5、6、9showed high resistance to P. versicolora and pCCA-3、4、7showed mediumresistance. The five high resistance lines showed higher toxicity than threesingle Cry3Aa gene lines with high resistance (pCC~(-1)1,53,84). The mortalityof adults feeded on these five lines was above60%at the third day andreached85%~100%at the fifth day, while the mortality feeded on three pCCseries was below50%at the third day and only60%~70%at the fifth day. In regards of H. cunea, seven lines (pCCA2pCCA7and pCCA9) exhibitedsimilar effectiveness as the single Bt line (pB29). The mortality of L4larvaereached100%at the7th~9th day. Only one line (pCCA1) showed anextremely low level of resistance. The mortality of L4larvae was7%and23%at the seventh day and the nineth day separately. pCC series showed notoxicity to H. cunea without Bt cry1Aa gene.
6. Feeding tests also showed either P. versicolora or H. cunea, L1larvaehad low tolerance to Bt toxic protein and died within2~3days. As for adult P.versicolora and L4larvae of H. cunea, tolerance increased obviously byextending the feed time to7~11days. From the demaged leaf area recordedby photos we can see that, even transgenic lines with midium and lowresistance level, all could perform a good protection of their leaves comparingto the control741plant.
7. The investigation of total frass and body lenghth of L4H. cunea larvaeshowed that the total frass of pCCA1was7~23times the weight of highinsect-resistance lines. Compared with that of the control741, only occupied25%of the contrast. About the body length, H. cunea larva feeded bynon-transgenic741poplar reached19mm; body length feeded by pCCA2-7,pCCA9and pB29was10~12mm, and that of low insect-resistance pCCA1 was only13mm. From the analysis of frass and body length illustrated thatthe transgenic lines had inhibited of growth and development of the larvea.
8. By Agrobacterium-mediated method and hygromycin (Hyg) asselectable marker, the genetic transformation of JuBa poplar with double Bt(Cry3Aa+Cry1Ac) genes was studied. The suitable differentiation mediumand the suitable Hyg concentration were deeply investigated as two keyfactors influencing the transformation efficiency. The best differentiationmedium was established as: MS+6BA0.25mg·L~(-1)+IBA0.1mg·L~(-1). The effectof different Hyg concentration (0mg·L~(-1)~15mg·L~(-1)) on leaves and shootswere investigated. Hyg5mg·L~(-1)was determined for the critical concentration.Through several successive transfer and selection in the medium containingHyg,4lines with stable Hyg resistance were obtained and named JB1, JB2,JB3and JB4.
9. The presence of the Cry1Ac gene and the Cry3Aa gene in these fourJuBa plants were detected by PCR analyses with special primers. The resultshowed that the target electrophoretic749bp bands of Cry1Ac gene and612bp bands of Cry3Aa gene in JB1, JB2, JB3and JB4appeared as the plasmidpositive template, while there was no target band in the control plant, preliminary indicating that the double Bt genes had been integrated into thegenome of JuBa poplar.
引文
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