| This topic of the research object--Wuta-tsai(Brassica campestris l.SSP.Chinensis(l.)Makino var.Rosularis Tsen et Lee)which are found in the Yangtze river area in our country,it mainly through the original cruciferous creat variation in long-term natural selection and come,like most plants,the leaves piece of Wuta-tsai mainly for the green and yellow,and combined with different geographical location and climate conditions present a different color.This plant for the biennial herbaceous plants [1].Because of the Wuta-tsai food nutrition is rich and delicious,cultivation are all over the world,and formed many local varieties.When We assess plants contain nutrition indicators,anthocyanin content is also an important index.Anthocyanins is widely in all kinds of plants have a variety of flavonoid compounds in organic matter,it has good water soluble.It decided to the color of plant fruit and flower petal.Anthocyanins can produce effect to the health of human body in function,mainly in the form of an antioxidant involved in the metabolism of human body,and improve skin vitality,promote the blood circulation,thus to prevent oxidation and hardening of the arteries,and the material also can effectively provide the amount of sugar sweets for the human body,for people with diabetes,the material is to be able to help themselves to the illness treatment [6,7].Both medicine and biology at present,for the use of anthocyanin and the research is in a state of more in-depth,many experts and scholars from medical need,committed to the artificial synthesis of anthocyanins by biotechnology.In the synthesis of raw materials,the most necessary is ANS enzyme reaction,this enzyme can contribute to the efficient synthesis of anthocyanins,smooth realization of design and color is have no color change [8].Now days,we have cloning to ANS gene from Arabidopsisthaliana,Brassica juncea,B.a lboglabra,biggest ampestris SSP.Rap cruciferous vegetables,but we haven't carried out for the Wuta-tsai substantive research ANS genes [4-14].The authors therefore in combination with the,on the basis of predecessors' research and successful experience.To study on Wuta-tsai of ANS cloning.at the same time for the final finished product gene in-depth discussion,the final construction of expression vector of justice and the theoretical basis for genetic transformation of Wuta-tsai molecular breeding.The main research results are as follows:(1)Through the Trisol method to extract RNA,we have five different vegetable varieties of RNA,and uv spectrophotometer to detect its purity,the results show that RNA OD260 / OD280 value around 1.7 mostly,agarose electrophoresis test also showed that the proposed RNA is pure.Then we will be five different vegetable varieties of RNA reverse transcribed into cDNA,elected by real-time fluorescent quantitative PCR in 5 different urivarieties of dishes varieties anthocyanins synthetase expression quantity highest varieties for subsequent testing.(2)We used NCBI find known crucifer ANS gene sequences,find out its amino acid sequence of conservative district,merger and primer design,using rt-pcr cloning of the gene,to get the length of 1077 bp,the gene encoding 359 amino acids,by NCBI database,the results show that with mustard,such as purple bolt homology is above 97%.(3)We will under the condition of 37 ? cloning at leaves patches on the carrier in the PGM-ANS cDNA fragments using the Sac ? EcoR and enzyme digestion,enzyme products from recycled after 1.0% agarose gel electrophoresis kit about 1077 bp fragment recycling,the segment with the Sac ? and EcoR ? enzyme of plant binary expression vector PBI121 with T4 DNA ligase from 16 ? connected under the condition of reaction,for the night.So the recombinant plasmid ANS fragment is inserted after 35 s promoter,TNOS before termination.Successful build ANS justice expression vector,named PBI121-ANS method of agrobacterium mediated transformation of tobacco,lay the foundation for follow-up study... |
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