向日葵HaLACS1的克隆、表达及酵母功能互补鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-1174

摘要/Abstract

摘要：

探究长链酰基辅酶A合成酶（long chain acyl-CoA synthetase,LACS）基因在向日葵（Helianthus annuus L.）油脂积累和逆境响应中的功能,为其在向日葵油脂合成和抗逆中的应用奠定基础。通过RT-PCR克隆得到向日葵HaLACS1的CDS序列,运用生物信息学方法分析HaLACS1的特点。利用实时荧光定量PCR（qRT-PCR）技术检测HaLACS1的组织表达特性及对NaCl、PEG和ABA的响应情况。通过构建GFP和HaLACS1的融合表达载体,转化拟南芥原生质体进行亚细胞定位分析。将HaLACS1转入酿酒酵母突变型菌株YB525中进行功能互补试验,并进行底物偏好性分析。结果显示,HaLACS1开放阅读框1 980 bp,编码659个氨基酸。蛋白进化树分析表明,HaLACS1与拟南芥AtLACS1和莴苣（Lactuca sativa）LsLACS1具有较高的相似性。拟南芥原生质体瞬时表达分析显示HaLACS1定位于内质网。实时荧光定量PCR结果显示,HaLACS1在所有组织中均有表达,但在种子发育的早期表达量较高,花次之。NaCl、PEG和ABA处理均能诱导HaLACS1在向日葵根、茎和叶中的表达。酵母功能互补试验证明HaLACS1蛋白具有LACS酶活性。底物偏好性分析表明HaLACS1偏好棕榈酸（C16：0）和油酸（C18：1）。表明HaLACS1与向日葵种子发育过程中的油脂积累和非生物胁迫响应相关。

关键词: 向日葵, HaLACS1, 亚细胞定位, 表达模式, 酵母功能互补

Abstract:

This work aims to investigate the functionality of long chain acyl-CoA synthetase（LACS）gene in Helianthus annuus L. in the oil accumulation and responses to abiotic stresses,for laying a foundation in the its oil synthesis and application in the tress responses. RT-PCR was applied to clone the CDS sequence of the HaLACS1,and bioinformatics method was to analyze the characteristics of the HaLACS1. Real-time quantitative PCR was employed to detect the expression patterns of HaLACS1 in different tissues and in responses to NaCl,PEG and ABA treatments. The fusion protein of GFP and HaLACS1 was constructed and transformed into Arabidopsis thaliana protoplasts for subcellular localization analysis. The HaLACS1 gene was transformed into a LACS-deficient yeast strain YB525 to examine the complementation effect and analyze its substrate. The results showed that the length of HaLACS1’s open reading frame was 1 980 bp,encoding 659 amino acids. The phylogenetic tree analysis demonstrated that HaLACS1 was highly similar with the AtLACS1 in A. thaliana and LsLACS1 in Lactuca sativa. The transient expression assays revealed that HaLACS1 was located in endoplasmic reticulum. qRT-PCR result showed that HaLACS1 was ubiquitously expressed in all tissues of H. annuus,but highly expressed in seeds at early developmental stage,followed in the flowers. The transcription levels of HaLACS1 were induced by NaCl,PEG and ABA in H. annuus roots,stems and leaves. Expression of HaLACS1 in the yeast strain YB525 demonstrated that HaLACS1 has LACS enzyme activity. Substrate preference analysis showed that HaLACS1 preferred palmitic acid（C16:0）and oleic acid（C18:1）. All these results suggest that HaLACS1 is related to H. annuus in response to abiotic stresses and oil accumulation in the process of seed development.

Key words: Helianthus annuus, HaLACS1, subcellular localization, expression patterns, yeast functional complementation

杨佳宝, 周至铭, 张展, 冯丽, 孙黎. 向日葵HaLACS1的克隆、表达及酵母功能互补鉴定[J]. 生物技术通报, 2022, 38(6): 147-156.

YANG Jia-bao, ZHOU Zhi-ming, ZHANG Zhan, FENG Li, SUN Li. Cloning,Expression of Helianthus annuus HaLACS1 Gene and Identification of Its Functional Complementation in Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2022, 38(6): 147-156.

图/表 11

表1 引物信息

Table 1 Information of primers used in this study

引物名称 Primer	引物序列 Primer sequence（5'-3'）	功能 Function
L1F	CCCAAGCTTATGGATAAGCTG- CAGTTTGC	HaLACS1克隆及酵母表达载体构建
L1R	CCGCTCGAGTTAAGACTTGTT- CCCGCCTA	HaLACS1克隆及酵母表达载体构建
qF	GAGCTCGAAAACGGAACGTG	qRT-PCR
qR	ATTGCAAGCCTCCATCGCTA	qRT-PCR
PF	CGGACTAGTATGGATAAGCT- GCAGTTT	pAN580-HaLACS1-eGFP载体构建
PR	CATGCCATGGAGACTTGTTC- CCGCCTA	pAN580-HaLACS1-eGFP载体构建
18S-F	CTACCACATCCAAGGAAGG- CAG	qRT-PCR
18S-R	CGACAGAAGGGACGAGTA- AACC	qRT-PCR

图1 向日葵HaLACS1的克隆（A）及染色体定位分析（B）

Fig. 1 Cloning（A）and chromosome location（B）of the HaLACS1 gene in H. annuus

图2 不同植物LACS1蛋白序列多重比对结果 At：拟南芥;Ha：向日葵;Ls：莴苣;Bn：甘蓝型油菜;Zm：玉米;Os：水稻

Fig. 2 Multiple alignment results of LACS1 proteins from different plants At：Arabidopsis thaliana. Ha：H. annuus L. Ls：Lactuca sativa. Bn：Brassica napus. Zm：Zea mays. Os：Oryza sativa

图3 向日葵HaLACS1启动子的顺式作用元件

Fig. 3 Cis-acting elements of the H. annuus HaLACS1 gene promoter

图4 向日葵HaLACS1蛋白与其他植物LACS1的系统进化树分析

Fig. 4 Phylogenetic relationships of the HaLACS1 protein in H. annuus with other plant LACS1

图5 HaLACS1在拟南芥原生质体中的亚细胞定位 A：HaLACS1的GFP（绿色）荧光;B：mCherry-HDEL（内质网marker）的红色荧光;C：明场;D：融合荧光

Fig. 5 Subcellular localization of the HaLACS1 protein in the protoplasts of Arabidopsis A：The green fluorescent signals of the HaLACS1 gene. B：Endoplasmic reticulum marker. C：Bright field. D：Merged

图6 HaLACS1在不同组织中的表达不同字母表示数值有显著差异（P<0.05）。下同

Fig. 6 Tissue expression analysis of the HaLACS1 gene Different letters indicate values are significantly different（P<0.05）. The same below

图7 向日葵在干旱（A）和盐（B）胁迫下不同组织中HaLACS1的表达分析

Fig. 7 Relative expressions of the HaLACS1 gene in H. annuus tissues under drought（A）and salt stresses（B）

图8 向日葵在ABA处理下不同组织中HaLACS1的表达分析

Fig. 8 Expression pattern of HaLACS1 in H. annuus tissues under ABA treatment

图9 重组酵母的生长状况

Fig. 9 Growth status of recombinant yeast cells

图10 重组酵母细胞的苏丹黑B染色

Fig. 10 Sudan black B staining of recombinant yeast cells **P<0.01

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