Study On Genetic Transformation And Exogenous Double Bt Genes Expression In Poplar

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^-11,53,84only 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 from3.26×10⁴to7.50×10⁵. The primary transcriptof Cry3Aa gene of pCC series and pCCA series were ranged from1.79×10⁸to6.05×10⁹. The results showed that the primary transcript of Cry3Aa gene（108¹09order of magnitude） was ten thousand times higher than that ofCry1Ac gene （104¹05order 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^-11,53,84and the CK741showed no color reaction. As to Cry3Aa protein, eightdouble Bt pCCA series and pCC^-11,53,84showed 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^-11,53,84). 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 the7th⁹th 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 within2³days. As for adult P.versicolora and L4larvae of H. cunea, tolerance increased obviously byextending the feed time to7¹1days. 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 pCCA1was7²3times 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 pB29was10¹2mm, 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^-115mg·L^-1) on leaves and shootswere investigated. Hyg5mg·L^-1was 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.