摘要
澳洲坚果果仁中含有丰富的不饱和脂肪酸(unsaturated fatty acid,UFA),其不饱和脂肪酸生物合成的分子机制还有待进一步解析。植物硬脂酰-酰基载体蛋白脱饱和酶(SAD)是脂肪酸生物合成途径中形成不饱和脂肪酸的关键酶。本研究以澳洲坚果(Macadamia integrifolia)光壳种为对象,通过PCR技术克隆获得澳洲坚果硬脂酰-酰基载体蛋白脱饱和酶基因(MiSAD),并对结构功能和表达模式进行了初步分析。结果显示,克隆获得5923 bp的MiSAD基因组DNA序列,该基因由3个外显子2个内含子组成,包含1191 bp的编码框,与公布的粗壳种MtSAD序列高度一致;编码396个氨基酸;MiSAD分子量为45.22 kDa,等电点为5.93,属于酰基-ACP脱饱和酶,是位于叶绿体或质体基质中的水溶性酶;高度保守的区域存在形成酶活位点的2个E-X-X-H二铁原子中心基序,N端和C端氨基酸序列差异较大;二级结构中以α-螺旋和无规则卷曲为主;三级结构预测中存在形成脂酰链结合部位的螺旋-转角-螺旋(HTH);与蒂罗花的同源性最高达94.2%,与其他物种的SAD同源性也都在80%以上;分子进化树显示与荷花进化关系较近,属于植物脂酰-ACP脱饱和酶。荧光定量PCR数据显示MiSAD在根、茎、叶、花、果中均有表达,其中叶片和果实中表达量较高,果实中的MiSAD表达量在开花后第100天左右达到最高,之后随着果实的成熟逐渐下降,呈现正态分布趋势。该研究为深入研究MiSAD在澳洲坚果果仁中不饱和脂肪酸生物合成的作用机制奠定基础。
Macadamia nuts contain abundant source of unsaturated fat acid(UFA),but the molecular mechanism of bio-synthesis of UFA remains to be further analyzed.Stearoyl acyl-carrier-protein desaturase catalyses the insertion of a double bond into saturated fatty acid bound in saturated acyl chains bound to ACP in higher plant,which is the key en-zyme in unsaturated fatty acid biological synthesis pathway.The Macadamia intergrifolia plant as research object,the full-length gDNA and cDNA of SAD gene encoding stearoyl acyl-carrier-protein desaturase were isolated from M.in-tegrifolia using PCR technique.The structure,function and expression pattern of MiSAD were preliminarily studied.The gDNA of SAD was 6947 bp and contained 3 exons and 2 introns.The open reading frame was 1191 bp and encoded 396 amino acids which was highly consistent with the public MtSAD sequence,and SAD was a water soluble enzyme with a total predicted molecular mass of 45.22 kDa and isoelectric point(pI)5.93 located in chloroplast or plasmids stroma.The amino acid sequences of SAD involved in active site showed highly conserved and significant difference in N-end and C-end.The highly conserved region contained 2 central motifs of E-X-X-H iron atoms which formed the active site of this enzyme.Theα-helix and random curl were predominate in the secondary structure.The he-lix-turning-helix was found in the prediction of tertiary structure for formation of fatty acyl chain binding site.Derived amino acid sequence showed the highest homology 94.2%with Telopea speciosissima,and the homology is above 80%in other species of SAD,close to Nelumbo nucifera in phylogentic tree.The SAD gene expression trends were basically identical by qRT-PCR.The SAD expression were found in root,stem,leaf,flower and macadamia nut,and the highest level was in nut.The expression of SAD gene increased about 90days after and reached the highest level at 100 days,then declined gradually in the following days during the macadamia nut maturation and presenting a normal distribution trend
作者
杨倩
杨子平
邹明宏
宋喜梅
万继锋
陈菁
罗炼芳
曾辉
YANG Qian;YANG Ziping;ZOU Minghong;SONG Ximei;WAN Jifeng;CHEN Jing;LUO Lianfang;ZENG Hui(South Subtropical Crops Research Institute,Chinese Academy of Tropical Agricultural Sciences,Zhanjiang,Guangdong 524091,China;Key Laboratory of Tropical Fruit Biology,Ministy of Agriculture and Rual Affairs Zhanjiang,Guangdong 524091,China;Key Laboratory of Tropical Crops Nutrition of Hainan Province,Zhanjiang,Guangdong 524091,China)
出处
《热带作物学报》
CSCD
北大核心
2023年第2期254-263,共10页
Chinese Journal of Tropical Crops
基金
海南省自然科学基金项目(No.321QN300)
广东省自然科学基金项目(No.2021A151501242)
中央级公益性科研院所基本科研业务费专项(No.1630062022002)。
关键词
澳洲坚果
硬脂酰-酰基载体蛋白脱饱和酶
基因克隆
表达分析
Macadamia integrifolia
stearoyl-acyl-carrier-protein desaturase(SAD)
gene cloning
expressing analysis
