Screening Aluminium-tolerant Genotype And Cloning The Aluminium Tolerance-regulated Gene Of Brassica Campestris L.ssp.Chinensis Makino

Brassica chinensis which belongs to cruciferous pakchoi subspecies, mainly distributes in southern China with abundant cultivars, but aluminum toxicity of the acid red soil in southern China has serious impact on its yield and quality.By cloning the aluminium resitance gene of Brassica chinensis and expecting to get new cultivars of Brassica chinensis with aluminium resitance by means of genetic engineering in this study.It not only sets a basis for genetic improvement of Brassica chinensis, but also is of great significance to use of red soil resources efficiently.This paper uses the five inbred lines of Aijiaohuang(97-3-2), Huangxinwu(003-27), Shanghaiqing(98-4-2), Huangyacai(97-8-6), Baimanjing(001-24) as experimental materials to screen most resistance aluminum genotype and least resistance aluminum genotype of Brassica chinensis, and adopts mutually complementary molecular technology cDNA-AFLP,Reverse Northern blot,RACE to clone the aluminium resistance gene of Brassica chinensis,and the research results as flowing:(1) It has confirmed that Aijiaohuang is the most resistance aluminum genotype and Huangyacai is the leat resistance aluminum genotype by different degree of aluminum stress for the five inbred lines of Brassica chinensis, and by means of the root relative elongation method and hematoxylin dyeing.(2)It uses the inbred line of Ajiaohuang and the inbred line of Huangyacai as experimental materials to form the RNA mix pond of seed root from different aluminum stress periods,and has screend out five aluminum resistance related TDFs by the technology of cDNA-AFLP,which are positive tested by reverse Northern blot.(3)Four TDFs has been sequenced successfully:H-A7T12485bp,its identities is100%with Oryza sativa Japonica Group cDNA,clone,and identities of its conding protein is100%with hypothetical protein of Oryza sativa Japonica Group; A-A8T17271bp,its identities is89%with ArabidopsisoxidoreductasNAD-bindingdomain-containing protein mRNA, and identities of its conding protein is85%with Arabidopsis FAD/NAD(P)-binding oxidoreductase; A-A19T12301bp, its identities is81%with Thellungiella mRNA, complete cds, clone,and identities of its conding protein is94%with ArabidopsisTIR class disease resistance protein identities;A-A10T8300bp, its identities is100%with Arabidopsis dihydrolipoamideS-acetyl transferase mRNA,and identities of its conding protein is100%with dihydrolipoamideS-acetyltransferase and it enhances resistance aluminum of Brassica chinensis through regulating cell physical metabolism by preliminary judgment.(4)By the means of RACE combining with Nest-PCR,the full length gene sequence of A-A10T81691bp has been gotten,including six ORFs, and the biggest ORF located in between299-1513, code405amino acids.the result of BLAST is that the full length gene of A-A10T8has100%identities with dihydrolipoamideS-acetyl transferase mRNA,and its coding protein has100%identities with dihydrolipoamideS-acetyl transferase.