摘要
油菜是我国种植面积最广的油料作物之一,提高油菜含油量和菜籽油品质是提高我国植物油市场竞争力的关键。因此,发掘优良品质基因,从基因水平上找到与提高油菜含油量和菜籽油品质的关键基因对种质资源的改良有重要意义。油菜溶血磷脂酸酰基转移酶基因(Lysophosphatidic Acid Acyltransferase, LPAAT)是已被证实的油脂合成的关键基因,分为质体型和微粒体型两类。目前对该基因的报道表明,该基因在不同物种和相同物种的不同品系中具有不同的底物偏好性。本研究以甘蓝型油菜(Brassica napus L.)B351未成熟种子为材料,分离BnLPAAT基因,进行功能初步研究。主要研究结果如下:
(1)从甘蓝型油菜B351中克隆得到3个BnLPAAT基因的ORF,pBnLPAAT、sBnLPAAT1、sBnLPAAT4,长度分别为1035 bp、1173 bp、1302 bp,生物信息学分析表明质体型pBnLPAAT与已知基因(GenBank登录号AF111161)同源性达99.9%;微粒体型sBnLPAAT1、sBnLPAAT4与已知基因(GenBank登录号Z95637)同源性达93.8%和85.3%。sBnLPAAT4与已知基因(GenBank登录号Z95637)相比有123 bp的重复区段。
(2)成功构建了BnLPAAT基因的酵母表达载体pPIC9K-pBnLPAAT、pPIC3.5K-sBnLPAAT1、pPIC3.5K-sBnLPAAT4,实现了BnLPAAT在毕赤酵母中的蛋白表达。对酵母脂肪酸组分进行分析表明,sBnLPAAT1、sBnLPAAT4对硬脂酸和油酸积累有促进作用
(3)构建了植物表达载体PBI121-pBnLPAAT,并成功转化拟南芥,为进一步分析该基因对植物体脂肪酸的积累的影响奠定了基础。
Rapeseed is one of the most widely cultivated oil crops in China. Inceasing the oil content and quality of seeds in Brassica napus is essential to improve our vegetable oil market competitiveness. Isolating the key genes which could improve the quality of rapeseed oil and oil content are important to enhance crop quality.
Lysophosphatidic Acid Acyltransferase (LPAAT) is proven to be the key enzyme of oil accumulation in unmatured seeds and is divided as plastidial type and the microsomal type.It is reported that LPAATs have different substrates preference either in different species or the same species in different strains. In this study, we isolated the LPAAT in immature Brassica napus L. B351. The main research results are as follows,
(1) Three sequences had been isolated from Brassica napus L. B351, the sequences are as follow,pBnLPAAT,sBnLPAAT1 sBnLPAAT4. The sequence length is 1035 bp, 1173 bp, 1302 bp respectively. Bioinformatics analysis showed that pBnLPAAT shares 99.9% identity with the gene deposits in GenBank(access number AF111161). sBnLPAAT1 and sBnLPAAT4 shares 93.8% and 85.3% identity with the gene deposits in GenBank(access number Z95637). sBnLPAAT4 have 123 bp repeats segment compared with the genes(GenBank access number Z95637).
(2) Pichia pastoris expression vectors pPIC9K-pBnLPAAT, pPIC3.5K-sBnLPAAT1 and pPIC3.5K-sBnLPAAT4 had been constructed and transferred Pichia pastoris.The protein was expressed successfully. Fatty acid components of Pichia pastoris analysis shows that, sBnLPAAT1 and sBnLPAAT4 can both improve the accumulation of Stearic acid and Oleic acid.
(3) A plant expression vector PBI121-pBnLPAAT had been constructed, and transferred Arabidopsis thaliana successful. Fatty acid components analysis will be carried out in the future.
引文
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