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Construction of Plant Expression Vector with bkt and crtR-B Gene and Its Genetic Transformations in Peanut and Maize

Author: XuJing
Tutor: QuYanYing；QinSong
School: Xinjiang Agricultural University
Course: Crop Genetics and Breeding
Keywords: Peanut (Arachis hypogaea L.) Maize（Zea mays L.） bkt gene crtR-B gene Vector construction Agrobacterium tumefaciens Genetic transformation Astaxanthin
CLC: S513
Type: Master's thesis
Year: 2006
Downloads: 149
Quote: 1
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Abstract


biosynthesis enzymes of astaxanthin which encoded by bkt gene and crtR-B gene.Peanut is one of theimportant oil and economic crops and there is no astaxanthin but muchβ-carotene in it.So,two genes weretransferred to peanut utilizing gene-engineering technique in the hope of providing some indexes for theproduction of large-scale astaxanthin using transgenic peanut. Plant expression vectorpCAMBIA1301-bkt-crtR with binary gene was constructed by inserting bkt gene and crtR-B gene obtainedby PCR method from vector pET-28a(+)-bkt and pET-28a(+)-crtR into vector pCAMBIA1301.Therecombination plasmid was introduced into Agrobacterium tumefaciens EHA105 by freezing-meltingtransformation method.Leaflets and apexes of embryo from two cultivars of Huayu 23 and Huayu 24, weretransformed with the strain of EHA105 harboring expression vector pCAMBIA1301-bkt-crtR.46 Kmresistance plants were obtained. PCR analysis confirmed that bkt gene and crtR-B gene had been integratedinto the peanut genome.Maize was an important grain and forage crop and there is much zeaxanthin in maize but noastaxanthin.The metabolic pathway of carotenoid biosynthesis shows that astaxanthin is synthesized byzeaxanthin with BKT enzyme. In order to add astaxanthin and improve the quality of maize,it is importantfor us to apply the genetic engineering to transfer bkt gene to maize.The Bkt gene expression cassette wasconstructed by replacing GUS gene in vector pBI221 with bkt gene obtained by PCR method from vectorpET-28a(+)-bkt, which contained the CaMV 35S promoter and NOS terminator.Plant expression vectorpCAMBIA1301-bkt was constructed by inserting bkt gene expression cassette into multiple cloning site invector pCAMBIA1301.The vector contained bkt gene and GUS gene.Bkt gene was transferred into maizeQi 319 mediated by Agrobacterium tumefaciens LBA4404 harboring expression vector pCAMBIA1301-bktand the resistant calli was obtained under hygromycin selection pressure .The results of GUS histochemicalassay indicated that GUS gene had been transferred into maize emberyogenic calli. And PCR analysisconfirmed that bkt gene had been integrated into maize calli.Reference searching results showed that it was the first time that bkt and crtR had been transferred topeanut and maize.Peanut (Arachis hypogaea L.)，Maize（Zea mays L.），bkt gene，crtR-B gene，Vectorconstruction，Agrobacterium tumefaciens，Genetic transformation，Astaxanthin

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CLC: > Agricultural Sciences > Crop > Cereal crops > Corn ( maize )
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