摘要
采用RT-PCR和RACE技术从油葵（Helianthus annuus L.）种子中克隆了DGAT基因的cDNA全长序列,命名为HaD1（GenBank登录号为HM 015632）.将HaD1与CaMV 35S组成型启动子融合,构建植物表达载体pBI-HaD1,通过根癌农杆菌介导转化烟草.对转基因植株进行GUS及PCR检测,同时采用气相色谱-质谱法（GC-MS）分析转基因烟草叶片中脂肪酸各成分的含量.结果表明：HaD1基因cDNA全长1 936 bp,最大开放阅读框为1 524 bp,编码507个氨基酸;推测的氨基酸序列与其它植物已报道的DGAT1基因的氨基酸序列一致性为70%~80%,具有DGAT1蛋白保守的二酰甘油结合基序HKWIVRHLYFP＂,因此HaD1基因属于DGAT1基因家族.GUS活性染色及PCR检测均证明外源HaD1整合到烟草基因组并成功表达.转基因烟草叶片脂肪酸含量测定发现,油酸、软脂酸和硬脂酸的含量得到提高,推测HaD1是植物油脂合成相关的重要基因.
Diacylglycerol acyltransferase（DGAT,EC 2.3.1.20） is a key enzyme in triacylglycerol（TAG） biosynthesis.In this work,the sequence of DGAT from sunflower（Helianthus annuus L.） was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE） which named HaD1（GenBank accession No.HM 015632）.The expression plasmid pBI-HaD1 was constructed by fusing the cDNA of HaD1 with the constitutive promoter CaMV 35S and introduced into tobacco（Nictiana tabacum） by Agrobacterium tumefaciens mediated transformation.Transgenic assays were performed using GUS activity and PCR analysis.At the same time,the composition and contents of fatty acids in leaves of transgenic tobacco were determined by GC-MS.The results showed that the length of HaD1 cDNA was 1 936 bp,which include an open reading frame（ORF） with 1 524 bp encoding 507 amino acids.The deduced amino acids sequence of HaD1 had 70%~80% identity with the reported DGAT1 proteins of other plants.A putative diacylglycerol-binding motif ＇HKWIVRHLYFP＇ wihich is unique to DGAT1,was observed in HaD1 protein indicated that HaD1 belongs to the DGAT1 gene family.The HaD1 gene was confirmed to be integrated into the genome of tobacco by GUS and PCR analysis.The leaves of transgenic tobacco contained higher amount of oleic acid（C18∶1）,palmitic acid（C16∶0） and stearic acid（C18∶0） than those of the untransformed plants.HaD1 gene is important in plant oil biosynthesis.
出处
《西北植物学报》
CAS
CSCD
北大核心
2011年第5期886-892,共7页
Acta Botanica Boreali-Occidentalia Sinica
基金
教育部"973"预研资助项目(104255)
关键词
油葵
二酰甘油酰基转移酶1
烟草
转基因
脂肪酸含量
sunflower
diacylglycerol acyltransferase 1
tobacco
transformation
fatty acids content
