生物技术通报 ›› 2022, Vol. 38 ›› Issue (6): 147-156.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1174
收稿日期:
2021-09-13
出版日期:
2022-06-26
发布日期:
2022-07-11
作者简介:
杨佳宝,男,硕士研究生,研究方向:植物油脂代谢调控;E-mail: 基金资助:
YANG Jia-bao1(), ZHOU Zhi-ming1, ZHANG Zhan2, FENG Li1, SUN Li1()
Received:
2021-09-13
Published:
2022-06-26
Online:
2022-07-11
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。
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.
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 | |
qF | GAGCTCGAAAACGGAACGTG | qRT-PCR |
qR | ATTGCAAGCCTCCATCGCTA | |
PF | CGGACTAGTATGGATAAGCT- GCAGTTT | pAN580-HaLACS1-eGFP载体构建 |
PR | CATGCCATGGAGACTTGTTC- CCGCCTA | |
18S-F | CTACCACATCCAAGGAAGG- CAG | qRT-PCR |
18S-R | CGACAGAAGGGACGAGTA- AACC |
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 | |
qF | GAGCTCGAAAACGGAACGTG | qRT-PCR |
qR | ATTGCAAGCCTCCATCGCTA | |
PF | CGGACTAGTATGGATAAGCT- GCAGTTT | pAN580-HaLACS1-eGFP载体构建 |
PR | CATGCCATGGAGACTTGTTC- CCGCCTA | |
18S-F | CTACCACATCCAAGGAAGG- CAG | qRT-PCR |
18S-R | CGACAGAAGGGACGAGTA- AACC |
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
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