山桐子IpSAD基因家族分析及功能鉴定

doi:10.12302/j.issn.1000-2006.202207034

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (6): 261-270.doi: 10.12302/j.issn.1000-2006.202207034

所属专题：南京林业大学120周年校庆特刊

山桐子IpSAD基因家族分析及功能鉴定

龚珏¹(), 毕浩¹, 杨雯露¹, 王东明², 王乐辉³, 姜渊忠¹, 马涛¹^,^*()

1.四川大学生命科学学院,四川成都 610064
2.四川省资阳市雁江区农业农村局,四川资阳 641300
3.四川丹桐科技有限公司,四川成都 610014

收稿日期:2022-07-21 修回日期:2022-09-16 出版日期:2022-11-30 发布日期:2022-11-24
基金资助:
国家重点研发计划(2021YFD2201100);国家自然科学基金项目(31922061)

Characterization of IpSAD gene family in Idesia polycarpa and functional identification

GONG Jue¹(), BI Hao¹, YANG Wenlu¹, WANG Dongming², WANG Lehui³, JIANG Yuanzhong¹, MA Tao¹^,^*()

1. School of Life Sciences, Sichuan University, Chengdu 610064, China
2. Ziyang Yanjiang District Agricultural and Rural Bureau of Sichuan Province, Ziyang 641300, China
3. Sichuan Dantong Technology Co., Ltd., Chengdu 610014, China

Received:2022-07-21 Revised:2022-09-16 Online:2022-11-30 Published:2022-11-24

摘要/Abstract

摘要：

【目的】硬脂酰-ACP Δ⁹脱氢酶(stearoyl-ACP Δ⁹ desaturase,SAD)是决定脂肪酸组成的关键酶,分离和鉴定木本油料植物山桐子(Idesia polycarpa)的SAD基因,可为遗传改良山桐子油的脂肪酸组成提供基因资源。【方法】使用 HMM 和 Blastp 鉴定山桐子全基因组中的SAD家族成员;利用MEGA X、MEME和PlantCare等在线软件对其蛋白质理化性质、系统发育关系、基因结构、保守基序和启动子顺式调控元件等进行分析;使用MCSCANX分析山桐子和毛果杨SAD基因之间的共线性关系;基于RNA-seq数据,分析IpSAD各成员的表达模式,通过病毒诱导的基因沉默技术(virus induced gene silencing,VIGS)快速验证它们的生物学功能。【结果】 ①在山桐子基因组中共鉴定到7个IpSAD基因(IpSAD1~7),多重序列比对结果显示各成员间序列相似度较高且结构域保守;②系统发育分析表明,IpSAD成员可以分为3个亚组,与拟南芥SAD家族的分类结果一致,拟南芥单不饱和油酸合成关键基因AtSSI2与山桐子IpSAD2、IpSAD3、IpSAD4的亲缘关系较近;③启动子区域顺势调控元件预测结果显示IpSAD基因的启动子含有光反应、脱落酸响应等顺式调控元件;④IpSAD基因按其组织表达模式可分为3类,第1类和第2类成员在大部分组织中表达量较低,而第3类成员在所有组织中表达量均较高。⑤沉默IpSAD基因使叶片的油酸含量显著降低,其中IpSAD3的沉默可使油酸含量下降76%。【结论】明确了IpSAD家族各成员的基本特征,确定了它们对于油酸合成的生物学功能,并为山桐子油脂生物合成调控机制研究奠定了基础。

关键词: 山桐子, 硬脂酰-ACP Δ⁹, 油酸, 病毒诱导基因沉默

Abstract:

【Objective】Stearoyl-ACP Δ⁹ desaturase (SAD) is a key enzyme that determines the composition of fatty acids. The whole-genome identification of Idesia polycarpa SAD genes can provide targets to improve fatty acid compositions by genetic methods.【Method】HMM and Blastp were used to identify IpSAD genes in the I. polycarpa genome. The gene structures, phylogenetic relationship, conserved motifs, physicochemical characters of IpSADs, and cis-regulatory element in these gene promoters were analyzed and predicted by MEGA X, MEME and PlantCare online software, respectively. The MCSCANX software was used to reveal the common linear relationship of SAD genes in I. polycarpa and P. trichocarpa. The tissue expression pattern of IpSAD genes was constructed according to RNA-seq, and the function of these genes was identified by a virus-induced gene silencing (VIGS) system.【Result】(1)A total of seven IpSAD genes (IpSAD1-7) were identified in the I. polycarpa genome, and these family members shared high sequence similarity and conserved domains. (2)Aphylogenetic tree indicated that IpSAD members were divided into three clades, which were consistent with the classification of the homologs in Arabidopsis thaliana. In particular,IpSAD2, IpSAD3 and IpSAD4 were closer to AtSSI2, which is the key enzyme of oleic acid biosynthesis. (3)The cis-regulatory elements in the promoters of IpSAD genes included elements of light and abscisic acid responses. (4)These IpSAD genes were divided into three categories according to their tissue expression patterns. The IpSAD genes in Classes Ⅰ and Ⅱ were expressed with a low level in most tissues, whereas Class Ⅲ genes showed a high level in all tested tissues. (5) Silencing IpSAD genes using VIGS significantly reduced the oleic acid content of leaves.Remarkably, the oleic acid content in IpSAD3 silenced leaves was decreased by up to 76%.【Conclusion】The characteristics of each IpSAD gene was clarified, and their functions in oleic acid synthesis were determined. Our findings can aid in the investigation of the biosynthesis mechanisms of oleic acid and total oil in I. polycarpa.

Key words: Idesia polycarpa, stearoyl-acyl carrier protein Δ⁹ desaturase (SAD), oleic acid, virus induced gene silencing(VIGS)

中图分类号:

S722.37
Q78

龚珏,毕浩,杨雯露,等. 山桐子IpSAD基因家族分析及功能鉴定[J]. 南京林业大学学报（自然科学版）, 2022, 46(6): 261-270.

GONG Jue, BI Hao, YANG Wenlu, WANG Dongming, WANG Lehui, JIANG Yuanzhong, MA Tao. Characterization of IpSAD gene family in Idesia polycarpa and functional identification[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(6): 261-270.DOI: 10.12302/j.issn.1000-2006.202207034.

图/表 9

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引物名称 primer name	序列 sequence	目的 purpose
SAD2-F	ATTCTCTAGAAGGCCTCCATGG GAAGCCAAATTACAGTGAGG
SAD2-R	CGGGACATGCCCGGGCCTCGAG GCTCTTCCACCAATGGCTAG
SAD3-F	ATTCTCTAGAAGGCCTCCATGGCGACTTGTTCATGCACCTCC
SAD3-R	CGGGACATGCCCGGGCCTCGAG ATGGCTCTCAAGCTCAATCC
SAD4-F	ATTCTCTAGAAGGCCTCCATGGATGAAACATGCAAAGATCCA
SAD4-R	CGGGACATGCCCGGGCCTCGAG AGCTCAATCCTTTTGTCTCT	VIGS
SAD5-F	CATG CCATGG TGGTGGAAATGGAATTCAAA
SAD5-R	CC CTCGAG CCTTCCCATCAATTCACAAC
SAD6-F	CATG CCATGG AGTGATGCACCAGTCTCGTC
SAD6-R	CC CTCGAG CCATGGAGAATTGGGCTAAG
SAD7-F	ATTCTCTAGAAGGCCTCCATGGCAGGGCTTCTTCAGTAATCA
SAD7-R	CGGGACATGCCCGGGCCTCGAG GATAACCTCTTGACGCTCCT
Q-IpSAD2-F	ATTTGGACAAGGGCATGGAC
Q-IpSAD2-R	ATGAACCCCAGGTAGGGACT
Q-IpSAD3-F	TCACATCATGGGCAATCTGG
Q-IpSAD3-R	TGGACTGTTCTCTGTTCCCG
Q-IpSAD4-F	AGCGTCTTGGAGTCTACACA	qRT-PCR
Q-IpSAD4-R	GAGGTAACCCGCAGACATAA
Q-IpSAD5-F	ACCTTCCTTCTCAATCTCGC
Q-IpSAD5-R	TTGTGGAGGCATAGAATGGG
Q-IpSAD6-F	ATGGCTCTCAAGCTGAACTC
Q-IpSAD6-R	GTGCCATGGAATGTGTCACT
Q-IpSAD7-F	TCTTATCGGGTCAGGAATGG
Q-IpSAD7-R	TCCACAATCTTGGTGTAGGC
UBQ-F	TTGACGGGAAAGACCATAAC
UBQ-R	CTTTCTCTTCTTGGCTCCAC

基因名 gene name	基因编号 gene ID	蛋白质信息 calculated protein information			理论等电点 theoretical pI
基因名 gene name	基因编号 gene ID	长度/aa length	分子质量/ku MW	亚细胞定位 subcellular localization	理论等电点 theoretical pI
IpSAD1	Idesia.03G00684	399	45.29	叶绿体	6.47
IpSAD2	Idesia.05G01258	396	45.29	叶绿体	5.99
IpSAD3	Idesia.07G00300	396	45.16	叶绿体	5.99
IpSAD4	Idesia.07G00301	398	45.23	叶绿体	6.11
IpSAD5	Idesia.18G01639	397	45.26	叶绿体	6.11
IpSAD6	Idesia.18G01640	386	44.47	叶绿体	6.64
IpSAD7	Idesia.20G00641	333	38.28	叶绿体	5.15

物种 species	来源 accession
拟南芥 Arabidopsis thaliana	At1g43800、At2g43710、At3g02610、At3g02620、At3g02630、At5g16230、At5g16240
毛果杨 Populus trichocarpa	Potri.010G179400、Potri.010G179500、Potri.013G127300、Potri.T034100、Potri.005G187600、Potri.008G077900
可可 Theobroma cacao	XP_007032081、XP_017974119、XP_017974120、XP_017974175、XP_017975512、XP_017977433、XP_017980831
油菜 Brassica nupus	BnaA01T0414100ZS、BnaA03T0212900ZS、BnaA03T0288900ZS,BnaA05T0039900ZS、BnaA10T0202500ZS、BnaA10T0202600ZS、BnaC01T0501000ZS、BnaC03T0251400ZS、BnaC03T0347000ZS,BnaC04T0043700ZS、BnaC09T0500900ZS、BnaC09T0501100ZS

山桐子IpSAD基因家族分析及功能鉴定

Characterization of IpSAD gene family in Idesia polycarpa and functional identification

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