Construction Of Insect-resistant And Color-leaf Gene Stacking Structure And Its Genetic Transformation In Populus Tomentosa

Populus tomentosa is an important fast-growing timber species and landscape tree species in China.This species generally exhibits characteristics of rapid growth and straight trunk,which make it play an important role in forestry economy,ecological construction and urban greening.However,the diseases and insects seriously affect the growth of P.tomentosa,while the monotonous green leaf color,to some extent,decreases its ornamental value.With the development of biotechnology,creating new varieties with both insect resistance and color-leaf characteristics by using genes stacking technology is significant to the germplasm innovation of P.tomentosa.The toxic proteins encoded by Btcry ? gene and Serine protease inhibitor Bbi gene are highly toxic to lepidoptera pests,while CmOr gene will cause leaf color change by controlling the carotenoid content.In this experiment,a gene stacking expression vector p CAMBIA1304-Btcry ?-IRES-CmOr-IRES-Luc was constructed using the IRES element,and an insect-resistance gene expression vector p CAMBIA1304-Bbi was constructed too.Both of them were separately transferred into P.tomentosa TC1521 by the Agrobacterium-mediated method,and a batch of positive transgenic plants were obtained through resistance screening and PCR identification.The expression of foreign genes were further detected by qRT-PCR and RT-PCR.In this study,it laid a foundation for subsequent physiological and biochemical index tests such as insect-resistance test and leaf pigment test.The main results of this study are as follows:1.Using p CAMBIA1304 plasmid as the backbone and IRES element as the mediate,a gene stacking structure “p CAMBIA1304-Btcry ?-IRES-CmOr-IRES-Luc” was designed and constructed,further PCR indentification showed that both Btcry ? and CmOr had been successfully integrated into the expression vector.2.The gene stacking structure p CAMBIA1304-Btcry ?-IRES-CmOr-IRES-Luc and the insect-resistant gene expression vector p CAMBIA1304-Bbi were respectively introduced into Agrobacterium GV3101 by freeze-thaw method.Subsequently genetic tranformation was performed in P.tomentosa TC1521 by Agrobacterium-mediated method.After plant tissue culture and hygromycin(Hyg)screening,40 root-resistant plantlets transformed with insect-resistance and color leaf bivalent genes and 18 root-resistant seedlings transformed with Bbi genes were respectively obtained.Finally,after PCR identification,8 bivalent gene-positive plants and 7 insect-resistant gene Bbi-positive plants were obtained.3.Then use RT-PCR and qRT-PCR techniques to further analysis of the expression of foreign genes in the above positive transgenic plants was conducted.The results showed that Btcry ? and CmOrgenes had been detected in 7 transgenic plants,and the relative expression level of foreign genes was significantly higher than that of wild-type plants(WT).Meanwhile,the transcripts of Bbi gene was also detected in the 2 transgenic Bbi plants using RT-PCR,which indicates that foreign genes have been integrated into the genome of P.tomentosa TC1521,and transcribed normally.4.The above 7 bivalent genes(Btcry ? and CmOr)transgenic lines and 2 Bbi insect-resistance transgenic lines were propagated,and a total of 24 bivalent gene transgenic plants and 16 Bbi insect-resistance plants were obtained.These transgenic plants were transplanted to pots with soil,and were moved to the greenhouse for cultivation.Current work laid the foundation for the subsequent insect-resistance test and leaf pigment analysis.