Antisense Transformation Of The Chalcome Isoenzyme Gene In Brassica Napus L.

Oilseed rape is one of the four major oil crops in the world and is one of the most important oil plants in China.In recent years,the yellow-seeded Brassica napus L.has attracted researchers' interest for good properties compared with black-seeded accession,such as thinner seed coat,lower seed coat pigment,and fiber content,higher seed oil and meal protein content,etc.Getting good character yellow-seeded rapeseed is one of the most important objectives for cultivator.Engineering genetics is useful to break blocks of different species and broad gene resources,therefore fasten breed process.However,the efficiency of target gene transgened into acceptor is very low.To date,21 Transparent Testa(TT)loci and a BAN locus have been identified in Arabidopsis thaliana,and many of the TT genes even have been cloned.However,the mechanism of yellow seed rape formation is not very clear.with both Arabidopsis thaliana and Brassica napus L.belone to Cruciferae,they are near in genetics.Achievements on the Transparent Testa trait in Arabidopsis thaliana are helpful for insight into the formation of the yellow seed color in B.napus.Chalcone isoenzyme(CHI,ECS.5.1.6)catalyzes 6'-hydroxylated chalcone into 5'-dihydroxyflavanone,CHI is an important enzyme in the early stages of flavonoid biosynthesisa, and is the first recognized enzyme.Mutation of CHI gene,induces A.thaliana seed coat color change from puce to yellow,and defined as TT5.Study on B.napus CHI gene is helpful to understand the molecular mechanism of the yellow seed color formation and high light on creating stable yellow-seeded lines by genetic engineering.In this paper,the hypocotyls of a B.napus cultivar "Huashuang No.4" were transformed by Agrobacterium tumefaciens carrying CHI gene antisense fragment and the reporter gene GUS was detected by histochemical pigmentation.The effects of kanamycin concentration,Agrobacterium culture methods and hormone combination in the pre-culture medium on transformation were analyzed.Some CHI antisense transgenic plants were identified through GUS histochemical pigmentation,PCR amplification and southern hybridization.Conclution as fellows:1.Effect of kanamycin(kan)concentration on transformation "Huashuang No.4" hypocotyls were cultivated in screening mediums with different kan concentrations after being infected and co-cultured with Agrobacterium tumefaciens,GUS reporter gene expression was detected by histochemical pigmentation 10 days later.Results showed that kan concentrateion have much influence on transformation and 15mg/L is best for transformation.2.Effect of Agrobacterium culture methods on transformationThe activated Agrobacterum are collected by centrifugation and subcultured with some different methods before infecting the hypocotyls.Reporter gene expression showed that subculture of Agrobacterium in MS with AS for some time after activating in LB medium promoted transformation.Obvious differences are observed among the six Agrobacterium culture methods adopted,of which S2-5 gave the best transformation result.To date,this methord has not been repored.However,our experiment results showed that it is important to better B.napus L.transgenic systerm.3.Effect of hormone combination in pre-culture medium on transformationThe hypocotyls were pre-cultured in mediums with different concentration of 2,4-D and 6BA before being infected with Agrobacterium.The results showed that combination of 2,4-D with 6BA in pre-culture have much more influence than others and the combination of 2.00mg/L 2,4-D and 0.25mg/L 6BA was best.4.Identification of the transgenic plantsThe regenerate transformend plants were detected with GUS histochemical pigmentation,PCR amplification of the foreign DNA segment and southern blot.Some transgenic plants were screened out.Southern-blot of genomic DNA showed that the antisense fragment of the CHI gene has integrated into the genome of the transgenic plants.Some of the transgenic oil seeds show slightly lightened color.But,there are no significant differences in other morphological characteristics compared with control.Through inhibiting of CHI expression with the antisense CHI fragment,the experiment results demonstrate that seed pigments acculumation was lowered,and indicates that flavonoid passway is realy involved in B.napus L.seed pigment acculumates.Also,it is helpful to explore B.napus L. seed pigment acculumation mechanism.