热带作物学报

澳洲坚果光壳种MiSAD的克隆与表达

PDF下载

杨倩 ¹^,²^,³ , 杨子平 ¹^,²^,³^,^* , 邹明宏 ¹ , 宋喜梅 ¹ , 万继锋 ¹ , 陈菁 ¹ , 罗炼芳 ¹ , 曾辉 ¹^,^*

热带作物学报 | 组学与生物技术 2023,44(2): 254-263

收起

热带作物学报 | 组学与生物技术 2023, 44(2): 254-263

澳洲坚果光壳种MiSAD的克隆与表达

全屏

杨倩¹^,²^,³, 杨子平¹^,²^,³^,^*, 邹明宏¹, 宋喜梅¹, 万继锋¹, 陈菁¹, 罗炼芳¹, 曾辉¹^,^*

作者信息

^1.中国热带农业科学院南亚热带作物研究所，广东湛江　524091

^2.农业农村部热带果树生物学重点实验室，广东湛江　524091

^3.海南省热带作物营养重点实验室，广东湛江　524091

杨倩（1985—），女，硕士，助理研究员，研究方向：作物资源与育种。

通讯作者:

^* 曾辉（ZENG Hui），E-mail: zenghui@catas.cn

杨子平（YANG Ziping），E-mail: yangziping302@163.com。

Cloning and Expressing Analysis of Stearoyl-acyl-carrier-protein Desaturase (SAD) from Macadamia intergrifolia

Qian YANG¹^,²^,³, Ziping YANG¹^,²^,³^,^*, Minghong ZOU¹, Ximei SONG¹, Jifeng WAN¹, Jing CHEN¹, Lianfang LUO¹, Hui ZENG¹^,^*

Affiliations

^1.South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, Guangdong 524091, China

^2.Key Laboratory of Tropical Fruit Biology, Ministy of Agriculture and Rual Affairs Zhanjiang, Guangdong 524091, China

^3.Key Laboratory of Tropical Crops Nutrition of Hainan Province, Zhanjiang, Guangdong 524091, China

出版时间: 2023-02-25 doi: 10.3969/j.issn.1000-2561.2023.02.004

文章导航

摘要

收起

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。

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

Abstract

收起

Macadamia nuts contain abundant source of unsaturated fat acid (UFA), but the molecular mechanism of biosynthesis 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 enzyme 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. integrifolia 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. The α-helix and random curl were predominate in the secondary structure. The helix-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. This study would build a foundation for elucidating the molecular mechanism of MiSAD in biosynthesis of unsaturated fatty acid in macadamia.

Key words

Macadamia integrifolia / stearoyl-acyl-carrier-protein desaturase (SAD) / gene cloning / expressing analysis

引用本文

杨倩, 杨子平, 邹明宏, 宋喜梅, 万继锋, 陈菁, 罗炼芳, 曾辉. 澳洲坚果光壳种MiSAD的克隆与表达. 热带作物学报, 2023 , 44 (2) : 254 -263 . DOI: 10.3969/j.issn.1000-2561.2023.02.004

Qian YANG, Ziping YANG, Minghong ZOU, Ximei SONG, Jifeng WAN, Jing CHEN, Lianfang LUO, Hui ZENG. Cloning and Expressing Analysis of Stearoyl-acyl-carrier-protein Desaturase (SAD) from Macadamia intergrifolia[J]. Chinese Journal of Tropical Crops, 2023 , 44 (2) : 254 -263 . DOI: 10.3969/j.issn.1000-2561.2023.02.004

基金

收起

海南省自然科学基金项目(321QN300)
广东省自然科学基金项目(2021A151501242)
中央级公益性科研院所基本科研业务费专项(1630062022002)

参考文献

收起

2023年第44卷第2期

PDF下载

引用本文

BibTeX

文章信息

doi: 10.3969/j.issn.1000-2561.2023.02.004

接收时间：2022-05-10
首发时间：2026-03-05
出版时间：2023-02-25

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2022-05-10
修回日期：2022-06-27

基金

海南省自然科学基金项目(321QN300)

广东省自然科学基金项目(2021A151501242)

中央级公益性科研院所基本科研业务费专项(1630062022002)

作者信息

^1.中国热带农业科学院南亚热带作物研究所，广东湛江　524091

^2.农业农村部热带果树生物学重点实验室，广东湛江　524091

^3.海南省热带作物营养重点实验室，广东湛江　524091

通讯作者:

^* 曾辉（ZENG Hui），E-mail: zenghui@catas.cn

杨子平（YANG Ziping），E-mail: yangziping302@163.com。

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/rdzwxb/CN/10.3969/j.issn.1000-2561.2023.02.004

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT