Screening And Identification Of Narrow Leaf Lines Based On Transgenic Rice

Transcription factor is upstream element which regulates expression of downstream gene.Recently,with the developing of molecular biotechnology and plant genetic engineering,the genetic transformation of plant is used to transfer transcription factors gene of the C₄ photosynthesis pathway into C₃ plant for the regulation of gene expression,and further improving of the photosynthesis efficiency in C₃ plant has become a research focus.Rice is an important economic food crop.In this study,the technology of Gateway is used to construct the expression vector of the target gene,and the tansgenic plants are obtained by Agrobacterium infection in Nipponbare.In this study,we are tested the agronomic traits and photosynthesis in the T0 and T1 transgenic plants.Futhermore,we are detected expression level of the endogenous genes related to auxin.On the basis of the results,it can be predicted the effects of target genes on plants.The main contents and results of this study were as follows:1.A total of 20 expression vectors with target genes are constructed by the Gateway cloning technology.The expression vectors can be used for genetic transformation.2.The expression vector is transfered into DH5αof E.coli by thermal excitation.Picking the DH5αsingle colony which appeared circle shape and milky colour on the LB medium containing spectinomycin,individual bacterial colonies PCR is used to test the target gene and hygromycin gene.Through alignment the sequence of the PCR products,it is verified that DH5αloaded an expression vector.3.The plasmid is extracted from the DH5αaccording to the protocol of plasmid extraction kit,and extraction is verified using agarose gel electrophoresis.4.The plasmid is transfered into Agrobacterium tumefaciens of AGL1 based on the liquid nitrogen freeze-thaw method.The expression vector is transformed into the callus of Nipponbare using Agrobacterium transformation.Before transplanted into the field,10plantlets are randomly selected to amplify for hygromycin gene and every sample presented positive electrophoresis band.40 plantlets of T0 generation are planted in the field.At 4-5 leaf stage,21 transgenic plants carrying GATA29 and 40 transgenic plants loading GBP19 appear obvious narrow-leaf trait.Others are without such a trait.5.T1 generation appears narrow-leaf trait segregation in GATA29 and GBP19respectively.And there is a separation ratio of Three to one for 100 plants using chi-square test in GATA29.So the narrow leaf is a dominant trait controlled by a single gene in GBP19.The target gene of GBP19 is detected in 100 plants of T1 rice.And the results show that 77 plants with narrow leaf traits have the target gene,while 23 plants without narrow leaf traits does not have the target gene.6.The main agronomic traits and photosynthesis are detected and analyzed for narrow-leaf plants in T1 rice.The results show that narrow-leave plants are significantly different from the wild type in plant height,leaf length,and leaf width,and there are no significant differences in grain size,morphology,and thousand-grain weight.Compared with wild type,the narrow-leave plants have significant differences in net photosynthetic efficiency,transpiration rate,intercellular CO₂ concentration,and stomatal conductance.7.qRT-PCR is used to detect the expression level of genes related to auxin biosynthesis,transport,and signal transduction in narrow-leaf plant carrying the gene of GBP19.The expression levels of the auxin-related genes PIN1,YUCCA1,LAX1,and IAA3all decrease,with the most decreasing for YUCCA1.It is predicted that the transcription factor of GBP19 could cause the narrow leaf trait through negatively regulating the YUCCA1 gene.