Study On The Exogenous Genes Expression Of The Insect-resistant Populus × Euramericana Cv. “74/76” And Deltocdes Cv. ‘55/56’ × Deltocdes Cv. ‘2KEN8’

With the development of genetic engineering,some resistant genes of biologies are transformed into forest tree genom,then reforming the characters and improving the quality of the forest trees.It has been widely applied.In order to define the interactions of genes and the structure of carriers in the multi gene transformation vectors effect on expressive stability and efficiency of the exogenous genes.The transgenic lines were select to do this experiment,for respectively,the three transgenic lines of Populus×euramericana cv.“74/76”with Cry1Ac-Cry3A-NTHK1genes,the three transgenic lines of Populus×euramericana cv.“74/76”with Cry1Ac-Cry3A-BADH genes and transgenic lines of deltocdes cv.‘55/56’×deltocdes cv.‘2KEN8’that transformed two kinds of transformed vectors with Bt Cry1Ac and Bt Cry3A genes,a total of eight lines,but their expression frame of structure are different.The exogenous DNA gene and toxic protein levels of all transgenic lines were detected at the same time,the photosynthesis indexs of each line of Populus×euramericana cv.“74/76”were detected,and a line was chosen randomly to do Tail-PCR detection,to find the position of exogenous target gene in the chromosome.The main results were as follows:1.The detection of PCR indicated that the target electophoretic bands of every tracgenic line was amplificated as same as the plasmid positive template,It indicated that the three exogenous genes exist stably in the three transgenic lines of Cry1Ac-Cry3A-NTHK1 of Populus×euramericana cv.“74/76”and three transgenic lines of Cry1Ac-Cry3A-BADH,so were the two kinds of vectors of eight trasgenic lines of deltocdes cv.‘55/56’×deltocdes cv.‘2KEN8’transformed Bt Cry1Ac and Bt Cry3A genes.2.Using fluorescence quantitative PCR to detect the expression of exogenous gene at the transcription level.The results showed that the transcript abundance of Cry1Ac genewas low,between 2.9×10³～7.2×10⁴,the transcript abundance of Cry3A gene was higher,4.2×10⁵～5.6×10⁷.Visiblely,these showed that the exogenous genes can express normally at the level of m RNA within Populus×euramericana cv.“74/76”and deltocdes cv.‘55/56’×deltocdes cv.‘2KEN8’.The transcript abundance of Cry3A was significantly higher than that of Cry1Ac gene between Populus×euramericana cv.“74/76”anddeltocdes cv.‘55/56’×deltocdes cv.‘2KEN8’.3.Bt toxic protein content of leaves upper branches of gm lines were detected to find the changing rules by ELISA in different months,and the Bt toxic protein content of leaves,phloem and xylem in upper,middle and lower of branches in August.The results showed that toxic protein of Cry1Ac and Cry3A expression were significantly different between all lines,and the toxic protein of Cry3A of each line was significantly higher than Cry1Ac,the quantity of Cry1Ac was very low.The expression rule of toxic protein of Cry1Ac of each carrier in the different month is that the gm lines of p1 and p2 carrier reached their peak in August,the toxic protein expression of p1 carrier was greater than that of p2 carrier,all the gm lines of p3 and p4 carrier reached their peak in September,the toxic protein expression of p4 carrier was greater than that of p3 carrier.The toxic protein expression of Cry3A was highest in August.The toxic protein expression of each carrier line increased slowly in June and July,and the toxic protein expression rised sharply in August and September,and then presented a clear downward trend.In August,the Bt toxic protein content of leaves,phloem and xylem in upper,middle and lower of branches,did not represent consistency.4.Comparing photosynthetic indexes of every transgenic line,through the analysis of net photosynthetic rate,transpiration rate and intercellular CO₂ concentration difference,the tests showed that the value of every photosynthetic indexes are different,but most of transgenic lines were significantly higher than the contral,and there were no significant difference between part of transgenic lines and the control.It showed that the exogenous genes did not effect on the photosynthetic capacity of plants.5.The Tail-PCR technology was used to detect the insert position of exogenous gene of transgenic line,and analysis of sequencing proved that the exogenous gene of No.2 of transgenic lines of Cry1Ac-Cry3A-NTHK1 of Populus×euramericana cv.“74/76”was on the 14th hromosomes of the receptor plants.Simultaneously,we found that the insertion of exogenous gene affected F-box protein domains.We found that there was not functional gene in the extension of the right border of the carrier when compared the nucleotide sequence of both sides of the insertion site,whereas there were starch branching enzyme gene sequences,11 COBRA-like protein gene sequence of Populus tomentosa Carr.and Populus trichocarpa hypothetical protein gene sequence in the left border.