Expression Study Of Exogenous Genes In Populus Nigra L.’s With Anti-Adversity Multiple Transgenic

Populus nigra L.’s with anti-adversity multiple transgenic uses multi-gene plant transformation vector with insect-resistant gene Bt Cry1Ac、Bt Cry3 A and salt-tolerant gene mtl D、BADH to genetic transform Populus nigra L.’s and obtain transgenic line by agrobacterium mediating method.This study used this as experimental material,through inspection of resistant insect to filtering out high insect-resistance hybrid,detecting the molecular characteristics of Bt gene of high insect-resistance hybrid,preliminary identifying the salt tolerance of part of high insect-resistance hybrid,to lay the foundation of cultivating the anti-adversity poplar varieties.(1)Feeding lepidopterous pests-Hyp Hantria cunea Drury and coleopteran pests-Plagiodera versicolora on 16 transgenic line which is l year old seedlings planted in the nursery to preliminarily filter highly 6 transgenic line which is No.1,No.7,No.9,No.11,No.12 and No.17 with efficient insect resistance that resulting in the lethality of the larvae of Hyp Hantria cunea Drury and Plagiodera versicolora is more than 90%.(2)Detect foreign gene of the 6 selected transgenic lines with high insect-resistance.PCR detection indicate the blade of the 6 selected transgenic strains are detected which had4 gene band with exogenousobjective.And the non-transgenic did not detect this situation.This result is initially proved 4 foreign gene have been inserted into the receptor poplar genome.(3)Fluorescence quantitative PCR was used to detect 6 transgenic strainsstrains and analysis the expression of two genes in different periods of the growing season which are in the blade of 6 selected transgenic strains.From Jun to September,the transcription levels of Cry1 Ac is on a downward trend.And the transcription levels of Cry3 A is on a rise trend.The transcription levels of Cry1 Ac and Cry3 A has a strong correlation.(4)Detect the Btviral protein expression on the blade of the 6 transgenic strains by ELISA.The transgenic strains both detected existing Btviral protein expression.And the control experiment did not detect it.From Jun to October,the quantity of viral protein expression of Cry1 Ac is generally on the rise.But it fell slightly in July.From Jun to October,the quantity of viral protein expression of Cry3 A is generally on a downward trend.But The quantity of viral protein expression of Cry3 A is significantly higher than the quantity of viral protein expression of Cry1 Ac.The expression of Cry1 Ac toxic protein was negatively correlated with the expression of Cry3 A toxic protein.(5)Feed different instars insect test on 6 selected strains.The result indicates the lethality of Hyp Hantria cunea Drury which is 1-2 years larvae is between 76.67%-96.67%on transgenic strains.The lethality of Hyp Hantria cunea Drury which is 3-4 years larvae is between 66.67%-93.33% on transgenic strains.The lethality of Hyp Hantria cunea Drurywhich is 5-6 years larvae is between 20.00%-88.89% on transgenic strains.These results are significantly higher than the non-transgenic control.The lethality of Plagiodera versicolora which is 1-2 years larvae is between 86.67%-100.00% on transgenic strains.The lethality of Plagiodera versicolora which is 3-4 years larvae is between70.00%-100.00% on transgenic strains.The lethality of Plagiodera versicolora which is5-6 years larvae is between 73.33%-93.33% on transgenic strains.These results are significantly higher than the non-transgenic control.(6)Use the 5 selected transgenic strains(No.1,No.7,No.9 No.12,No.17)and non-transgenic control CK tissue culture seedling to test the potted salt tolerance.Comprehensive evaluating the salt tolerance,the sort of the salt tolerance among transgenic and non-transgenic controls is No.17>No.7>No.1>CK>No.9>No.12.(7)Detect the foreign gene products of transgenic strains under the salt stress.The results showed that the content of toxic protein in each transgenic line was significantly higher than that under clear water.The non-transgenic controls did not detect toxic proteins.And the betaine and mannitol which are the products of gene BADH and mtl Dwere detected in both transgenic and non-transgenic strains.Under the salt stress,the content of betaine and mannitol in each strain increased compared with water.However,the difference in betaine and mannitol content between the transgenic and non-transgenic control leaves was not significant.