磷酸泛酰巯基乙胺基转移酶基因过量表达对集胞藻PCC6803脂肪酸合成的影响

doi:10.3969/j.issn.1004-1524.2022.06.22

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1316-1325.DOI: 10.3969/j.issn.1004-1524.2022.06.22

磷酸泛酰巯基乙胺基转移酶基因过量表达对集胞藻PCC6803脂肪酸合成的影响

李燕乐¹(), 钟怀荣², 宣宁², 张燕², 陈高²^,^*(), 季祥¹^,^*()

1.内蒙古科技大学生命科学与技术学院,内蒙古自治区生物质能源化利用重点实验室,内蒙古包头 014010
2.山东省农业科学院生物技术研究中心,山东省作物遗传改良与生态生理重点实验室,山东济南 250100

收稿日期:2021-01-29 出版日期:2022-06-25 发布日期:2022-06-30
通讯作者: 陈高,季祥
作者简介:季祥,E-mail: jixiang@imust.cn
*陈高,E-mail: gxchen001@hotmail.com;
李燕乐（1994—）,女,内蒙古乌兰察布人,硕士研究生,主要从事藻类分子生物学研究。E-mail: ll525611@163.com
基金资助:
内蒙古自然科学基金(2019MS02024);国家重点研发计划政府间国际科技创新合作/港澳台科技创新合作重点专项(2018YFE0108600);山东省重点研究计划（公益性科技攻关类）(2018GSF121019);山东省重点研究计划（公益性科技攻关类）(2019GSF107098)

Effects of over expression of phosphopantetheinyl transferases gene on fatty acid synthesis in Synechocystis sp. PCC6803

LI Yanle¹(), ZHONG Huairong², XUAN Ning², ZHANG Yan², CHEN Gao²^,^*(), JI Xiang¹^,^*()

1. Inner Mongolia Key Laboratory of Biomass-Energy Conversion, School of Life and Technology, Inner Mongolia University of Science and Technology, Baotou 014010, Inner Mongolia, China
2. Shandong Key Laboratory of Crop Genetics and Breeding and Ecological Physiology, Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China

Received:2021-01-29 Online:2022-06-25 Published:2022-06-30
Contact: CHEN Gao,JI Xiang

摘要/Abstract

摘要：

磷酸泛酰巯基乙胺基转移酶（phosphopantetheinyl transferases,PPTase）是细菌脂肪酸合成中的关键酶。本研究利用同源重组手段构建集胞藻PPTase基因（slr0495）过量表达重组质粒,在集胞藻PCC6803中进行表达研究,并在DNA和RNA水平进行验证,利用气相色谱-质谱联用仪（GC-MS）检测不同条件下突变藻株脂肪酸组分及含量。结果表明：在温度为30 ℃、光照强度为50 μmol ·m^-2· s^-1培养条件下,过量表达slr0495基因突变藻株中C12:0、C16:0和C18:0的含量分别为1. 31 mg · g^-1、8.07 mg · g^-1和1.35 mg · g^-1,比野生型藻株分别提高了23.58%、25.31%和13.45%;在温度为20 ℃、50% NaNO₃浓度、光照强度为50 μmol · m^-2· s^-1培养条件下,与野生型相比,突变藻株中C16:0和C18:0含量分别提高了44.71%和41.51%。以上结果表明,过表达slr0495基因增加了集胞藻PCC6803中长链饱和脂肪酸的含量,并且在低温（20 ℃）和缺氮（50% NaNO₃）双重胁迫培养条件下中长链饱和脂肪酸含量得到进一步提高。本研究为探究slr0495基因功能以及在逆境中基因产生的应激反应提供了理论依据,为微藻高产多不饱和脂肪酸代谢研究奠定了基础。

关键词: 集胞藻PCC6803, 磷酸泛酰巯基乙胺基转移酶, 脂肪酸, 同源重组

Abstract:

Phosphopantothenyl transferase (PPTase) is a key enzyme in fatty acid synthesis in bacteria. In this study, the homologous recombinant plasmid slr0495⁽⁺⁾ was successfully constructed for overexpressing the PPTase (slr0495) gene and transformed into wild-type Synechocystis sp. PCC6803. The results showed that under the culture conditions of 30 ℃, 50 μmol·m^-2·s^-1, the contents of C12:0, C16:0 and C18: 0 in wild type Synechocystis sp. were 1.06 mg·g^-1, 6.44 mg·g^-1, 1.19 mg·g^-1,while the C12:0, C16:0and C18:0 contents of mutant strains were 23.58%, 25.31% and 13.45% higher than those of wild type strains, respectively. Under the culture conditions of 20 ℃, 50% NaNO₃ and 50 μmol·m^-2·s^-1 light intensity, compared with the wild type, the contents of C16:0 and C18:0 in mutant strains overexpressing slr0495 gene increased by 44.71% and 41.51%, respectively. These above results indicated that overexpression of slr0495 gene increased the contents of saturated long-chain fatty acids in Synechocystis sp. PCC6803, and the contents of medium-long-chain saturated fatty acids were further improved under the conditions of low temperature (20 ℃) and nitrogen deficiency (50% NaNO₃). This study not only provides a theoretical basis for further exploring the function of slr0495 and the stress response of this gene in adversity but also lays the foundation for further exploring the high-yield polyunsaturated fatty acids of microalgae.

Key words: Synechocystis sp. PCC6803, phosphopantetheinyl transferases, polyunsaturated fatty acids, homologous recombination

中图分类号:

Q786

李燕乐, 钟怀荣, 宣宁, 张燕, 陈高, 季祥. 磷酸泛酰巯基乙胺基转移酶基因过量表达对集胞藻PCC6803脂肪酸合成的影响[J]. 浙江农业学报, 2022, 34(6): 1316-1325.

LI Yanle, ZHONG Huairong, XUAN Ning, ZHANG Yan, CHEN Gao, JI Xiang. Effects of over expression of phosphopantetheinyl transferases gene on fatty acid synthesis in Synechocystis sp. PCC6803[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1316-1325.

图/表 9

图1 集胞藻PCC6803脂肪酸合成途径

Fig.1 Fatty acid synthesis pathway of Synechocystis sp. PCC6803

表1 引物序列

Table 1 Primer used in this study

引物 Primers	序列 Primer sequences（5'→3'）
Pcpc560-F	AATGTCGACACCTGTAGAGAAGAGTCCC
Pcpc560-R	TGAATTAATCTCCTACTTGAC
slr0646U-F	CGCCTCGAGACGGCAATTGGTTATCACT
slr0646U-R	CCAGTCGACACGCTGCAATTTGACCTAT
slr0646D-F	ATACCGCGGAAACAACGCCGCTGACGAA
slr0646D-R	GGCGAGCTCTACAAGAAGCAGAGGAGTT
slr0495-F	AGTAGGAGATTAATTCAATGCTCCCCCAGCCCCAAA
slr0495-Flag-R	GGCGAATTCCTACTTATCGTCGTCATCCTTGTAATC
slr0495-Flag-RT-F	GATACAAACCATTGGCGA
slr0495-Flag-RT-R	TTGGAACTAAGTAACGGG

图2 slr0646UD 同源重组平台构建及检测 A,集胞藻PCC6803基因组DNA电泳结果;B,slr0646UD DNA水平PCR扩增结果;C,slr0646UD 同源重组平台质粒构建图;WT,集胞藻PCC6803。

Fig. 2 Construction process and detection of homologous recombination platform slr0646UD A, Agorase gel electrophoresis of genome from Synechocystis sp. PCC6803; B, The identification of slr0646UD platform in DNA level; C, Construction map of slr0646UD homologous recombination platform; WT, Synechocystis sp. PCC6803.

图3 突变株slr0495(+) DAN和RNA水平鉴定 A,同源重组质粒slr0495 (+)构建图谱;B,突变株slr0495(+) PCR扩增检测图;C,突变株slr0495(+) RNA水平鉴定;WT,集胞藻PCC6803;M,突变株slr0495(+)。

Fig. 3 The identification of slr0495(+) mutant in DNA and RNA level A, Construction of homologous recombinant plasmid slr0495(+); B, PCR amplification of mutant slr0495 (+); C, The identification of slr0495(+) mutant in RNA level; WT, Synechocystis sp. PCC6803; M, Mutant strain of slr0495(+).

图4 温度及氮浓度对藻株生长速率的影响 A,30 ℃、50 μmol· m-2·s-1、不同氮浓度条件下的生长曲线;B,20 ℃、50 μmol·m-2·s-1、不同氮浓度条件下的生长曲线;WT,集胞藻PCC6803;M,突变株slr0495(+)。

Fig. 4 Effects of temperature and nitrogen concentration on growth rate of algae A, Growth curves at 30 ℃, 50 μmol·m-2·s-1 and different nitrogen concentrations; B, Growth curves at 20 ℃,50 μmol·m-2·s-1 and different nitrogen concentrations; WT, Synechocystis sp. PCC6803; M, The mutant strain of slr0495(+).

图5 叶绿素a含量 A,30 ℃、50 μmol·m-2·s-1不同氮浓度培养条件下叶绿素a含量;B,20 ℃、50 μmol·m-2·s-1不同氮浓度培养条件下类叶绿素a含量;WT,集胞藻PCC6803;M,突变株slr0495(+)。

Fig. 5 Content of chlorophyll a A, Chlorophyll a contents under different nitrogen concentrations at 30 ℃, 50 μmol·m-2·s-1; B, Chlorophyll a content under different nitrogen concentrations at 20 ℃, 50 μmol·m-2·s-1; WT, Synechocystis sp. PCC6803; M, Mutant strain of slr0495(+).

图6 类胡萝卜素含量 A,30 ℃、50 μmol·m-2·s-1不同氮浓度培养条件下类胡萝卜素含量;B,20 ℃、50 μmol·m-2·s-1不同氮浓度培养条件下胡萝卜素含量。

Fig. 6 Content of carotenoids A, Carotenoid contents under different nitrogen concentrations at 30 ℃, 50 μmol·m-2·s-1; B, Carotenoid contents under different nitrogen concentrations at 20 ℃, 50 μmol·m-2·s-1.

表2 不同胁迫条件下野生型和突变株slr0495(+)脂肪酸组分的含量

Table 2 Fatty acid contents of wild type and slr0495(+) mutants under different conditions mg·g-1

脂肪酸Fatty acid	WT 30 ℃ 100% N 50% N		M 30 ℃ 100% N 50% N		WT 20 ℃ 100% N 50% N		M 20 ℃ 100% N 50% N
TFA	13.42 ± 0.63	15.25 ± 0.47	19.69 ± 1.02	22.43 ± 1.17	24.52 ±1.12	29.44 ± 1.83	22.31 ± 1.47	30.44 ± 1.75
C11:0	6.59 ± 0.27	6.96 ± 0.32	3.27 ± 0.17	3.85 ± 0.23	6.79 ± 0.43	5.96 ± 0.21	5.66 ± 0.32	6.48 ± 0.49
C12:0	1.06 ± 0.09	1.24 ± 0.12	1.31 ± 0.16	1.43 ± 0.11	1.53 ± 0.07	1.57 ± 0.13	1.62 ± 0.22	1.89 ± 0.16
C16:0	6.44 ± 0.32	6.75 ± 0.27	8.07 ± 0.23	8.97 ± 0.12	7.94 ± 0.65	8.41 ± 0.21	9.45 ± 0.39	12.17 ± 0.63
C18:0	1.19 ± 0.07	1.24 ± 0.23	1.35 ± 0.03	1.41± 0.07	1.54 ± 0.13	1.59 ± 0.11	1.71 ± 0.15	2.25± 0.07
C18:3	3.02 ± 0.27	3.27 ± 0.21	3.61 ± 0.15	3.93 ± 0.23	0.37 ± 0.07	0.56 ± 0.07	4.01 ± 0.12	4.34 ± 0.21

图7 不同温度及氮浓度条件下藻株的Fv/Fm值

Fig. 7 Fv/Fm values of algal strains at different temperatures and nitrogen concentrations

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磷酸泛酰巯基乙胺基转移酶基因过量表达对集胞藻PCC6803脂肪酸合成的影响

Effects of over expression of phosphopantetheinyl transferases gene on fatty acid synthesis in Synechocystis sp. PCC6803

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