BnaLPAT2-A07 Gene Regulates Oil Content and Fatty Acid Composition in Brassica napus Seeds

doi:10.7668/hbnxb.20194849

Abstract

Abstract:

To investigate the function of lysophosphatidicacid acyltransferase 2(LPAT2)in Brassica napus,from which one copy(A07)of BnaLPAT2 was cloned by PCR.we constructed the overexpression vectors p35S∷BnaLPAT2-A07 and the seed-specific expression vector pNapin∷BnaLPAT2-A07,and by utilizing Agrobacterium-mediated genetic transformation method,obtained a total of 15 and 11 transgenic Brassica napus cv.Zhongshuang 6 respectively by PCR positive detection.Real-time Quantitative PCR(qRT-PCR)showed that the transcript levels of BnaLPAT2-A07 in most tissues of T₃ overexpressed rapeseed were higher than that of CK.However,in the seed-specific expression tissues of T₃ transgenic rapeseed,the BnaLPAT2-A07 genes were strongly expressed in the development and maturation stages of silique.Soxhlet extraction results showed that the oil content in the transgenic seeds driven by the 35S or Napin promoter accumulated 1.39 and 2.36 percentage point more oil than control seeds,respectively.The fatty acid components of transgenic rape were detected by gas chromatography.Compared with CK,the content of linolenic acid was increased by 3.13 and 1.47 percentage point,respectively.Taken together,the BnaLPAT2-A07 could promote seed oil synthesis,however,the specific selection function of BnaLPAT2-A07 for linolenic acid needs to be further verified.

Key words: Brassica napus, Over expression, BnaLPAT2-A07, Fatty acid analysis, Oil content

QIN Yi, LIU Yong, XIONG Xinghua. BnaLPAT2-A07 Gene Regulates Oil Content and Fatty Acid Composition in Brassica napus Seeds[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 45-52. doi: 10.7668/hbnxb.20194849.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194849

http://www.hbnxb.net/EN/Y2025/V40/I1/45

Figures/Tables 7

References 34

[1]	高华妮. 拟南芥甘油-3-磷酸酰基转移酶(GPAT)参与表皮脂类合成的功能解析[D]. 武汉: 湖北大学, 2022.doi:10.27130/d.cnki.ghubu.2022.000387.
	Gao H N. Functional analysis of Arabidopsis GPAT involved in surface lipid synthesis[D]. Wuhan: Hubei University, 2022.
[2]	郝翠翠, 梁成伟, 石蕾, 李昊远, 陈明娜, 潘丽娟, 王通, 王冕, 禹山林, 陈娜, 迟晓元. 花生甘油-3-磷酸酰基转移酶(GPAT)基因的克隆及表达分析[J]. 花生学报, 2018, 47(1):1-10,18.doi:10.14001/j.issn.1002-4093.2018.01.001.
	Hao C C, Liang C W, Shi L, Li H Y, Chen M N, Pan L J, Wang T, Wang M, Yu S L, Chen N, Chi X Y. Cloning and expression analysis of glycerol-3-phosphate acyltransferase(GPAT)genes in peanut[J]. Journal of Peanut Science, 2018, 47(1):1-10,18.
[3]	尹航, 袁岚瑛, 俞凯, 章丽, 石慧, 王何瑜, 龚一富. 三角褐指藻GPAT生物信息学及表达差异分析[J]. 核农学报, 2019, 33(8):1483-1489.doi:10.11869/j.issn.100-8551.2019.08.1483.
	Yin H, Yuan L Y, Yu K, Zhang L, Shi H, Wang H Y, Gong Y F. Bioinformatics and expression differences of GPAT in Phaeodactylum tricornutum[J]. Journal of Nuclear Agricultural Sciences, 2019, 33(8):1483-1489.
[4]	陶芬芳, 邢蔓, 岳宁燕, 邬贤梦. 植物三酰甘油合成相关基因研究进展[J]. 作物研究, 2017, 31(3):330-336.doi:10.16848/j.cnki.issn.1001-5280.2017.03.27.
	Tao F F, Xing M, Yue N Y, Wu X M. Research advances of genes related to plant triacylglycerol synthesis[J]. Crop Research, 2017, 31(3):330-336.
[5]	Jako C, Kumar A, Wei Y D, Zou J T, Barton D L, Giblin E M, Covello P S, Taylor D C. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight[J]. Plant Physiology, 2001, 126(2):861-874.doi:10.1104/pp.126.2.861. pmid: 11402213
[6]	汪慧慧, 胡蓉, 岳宁燕, 官春云, 熊兴华. 植物LPATs基因研究进展[J]. 分子植物育种, 2020, 18(4):1138-1143.doi:10.13271/j.mpb.018.001138.
	Wang H H, Hu R, Yue N Y, Guan C Y, Xiong X H. Research progress on lysophosphatidic acid acyltransferase(LPATs)gene in plants[J]. Molecular Plant Breeding, 2020, 18(4):1138-1143.
[7]	叶冠华. 基于Kennedy路径关键酶基因调控莱茵衣藻油脂代谢的研究[D]. 深圳: 深圳大学, 2018.doi:10.13271/j.mpb.018.001138.
	Ye G H. Study on the regulation of lipid metabolism of Chlamydomonas reinhardtii based on Kennedy pathway key enzyme genes[D]. Shenzhen: Shenzhen University, 2018.
[8]	Gao Y, Sun Y, Gao H L, Chen Y, Wang X Q, Xue J N, Jia X Y, Li R Z. Ectopic overexpression of a type-Ⅱ DGAT(CeDGAT2-2)derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves[J]. Biotechnology for Biofuels, 2021, 14(1):76.doi:10.1186/s13068-021-01928-8. pmid: 33757551
[9]	杨涔, 陈丽玉, 廖春梅, 孔凡江. 大豆DGAT1/2基因表达分析和敲除靶点设计[J]. 大豆科学, 2022, 41(4):438-447.doi:10.11861/j.issn.1000-9841.2022.04.0438.
	Yang C, Chen L Y, Liao C M, Kong F J. Gene target design and functional analysis of soybean DGAT1/2 genes[J]. Soybean Science, 2022, 41(4):438-447.
[10]	Singer S D, Chen G Q, Mietkiewska E, Tomasi P, Jayawardhane K, Dyer J M, Weselake R J. Arabidopsis GPAT9 contributes to synthesis of intracellular glycerolipids but not surface lipids[J]. Journal of Experimental Botany, 2016, 67(15):4627-4638.doi:10.1093/jxb/erw242. pmid: 27325892
[11]	刘勇, 刘聪, 戴晨曦, 熊兴华, 官春云. 甘蓝型油菜GPAT6基因在酵母和拟南芥中的表达分析[J]. 分子植物育种, 2023, 21(7):2125-2131.doi:10.13271/j.mpb.021.002125.
	Liu Y, Liu C, Dai C X, Xiong X H, Guan C Y. Expression analysis in yeast and Arabidopsis of bn GPAT6 gene in Brassica napus[J]. Molecular Plant Breeding, 2023, 21(7):2125-2131.
[12]	刘铭宇. 过表达与三酰甘油有关的几个基因提高拟南芥种子的含油量[D]. 武汉: 华中农业大学, 2020.doi:10.27158/d.cnki.ghznu.2020.000960.
	Liu M Y. Overexpression of several genes related to triacylglycerol improves the oil content of Arabidopsis seeds[D]. Wuhan: Huazhong Agricultural University, 2020.
[13]	皮广静. 花生油脂合成酰基转移酶基因表达与籽仁含油量相关性分析[D]. 泰安: 山东农业大学, 2017.doi:10.7666/d.D01212031.
	Pi G J. Correlation analysis between the expression of acyltransferase gene in peanut oil synthesis and oil content in seed[D]. Taian: Shandong Agricultural University, 2017.
[14]	胡蓉, 汪慧慧, 刘勇, 熊兴华. 甘蓝型油菜BnaLPAT2基因在拟南芥中的种子特异性表达及过表达分析[J]. 湖南农业大学学报(自然科学版), 2021, 47(1):17-22.doi:10.13331/j.cnki.jhau.2021.01.003.
	Hu R, Wang H H, Liu Y, Xiong X H. Analysis of seed-specific expression and overexpression of BnaLPAT2 gene in Arabidopsis thaliana[J]. Journal of Hunan Agricultural University(Natural Sciences), 2021, 47(1):17-22.
[15]	柏杨, 章文华. 二酰甘油从头合成途径的关键酶及其功能[J]. 植物生理学报, 2018, 54(12):1763-1773.doi:10.13592/j.cnki.ppj.2018.0422.
	Bai Y, Zhang W H. Key enzymes for de novo synthesis of diacylglycerol in plant cells[J]. Plant Physiology Journal, 2018, 54(12):1763-1773.
[16]	董芳, 迟晓元, 杨庆利, 陈娜, 潘丽娟, 陈明娜, 和亚男, 杨珍, 王通, 王冕, 禹山林. 三个花生溶血磷脂酸酰基转移酶基因的克隆与序列分析[J]. 花生学报, 2013, 42(1):1-11.doi:10.14001/j.issn.1002-4093.2013.01.004.
	Dong F, Chi X Y, Yang Q L, Chen N, Pan L J, Chen M N, He Y N, Yang Z, Wang T, Wang M, Yu S L. Cloning and sequence analysis of three lysophosphatidic acid acyltransferase(LPAT)encoding genes in peanut[J]. Journal of Peanut Science, 2013, 42(1):1-11.
[17]	丁检, 吴双, 蔡彩平, 郭旺珍. 棉花溶血磷酸酯酰转移酶(LPAT)家族基因的发掘和表达分析[J]. 作物学报, 2015, 41(3):378-385.doi:10.3724/SP.J.1006.2015.00378.
	Ding J, Wu S, Cai C P, Guo W Z. Genome-wide identification of lysophosphatidic acid acyltransferase gene family and their expression analysis in cotton[J]. Acta Agronomica Sinica, 2015, 41(3):378-385.
[18]	尹永泰. 甘蓝型油菜溶血磷脂酰基转移酶家族基因的克隆与表达[D]. 武汉: 华中科技大学, 2016.doi:10.7666/d.D01078461.
	Yin Y T. Cloning and expression of lysophosphatidis acyltransferase family genes in Brassica napus[D]. Wuhan: Huazhong University of Science and Technology, 2016.
[19]	Misra N, Panda P K, Parida B K. Genome-wide identification and evolutionary analysis of algal LPAT genes involved in TAG biosynthesis using bioinformatic approaches[J]. Molecular Biology Reports, 2014, 41(12):8319-8332.doi:10.1007/s11033-014-3733-1. pmid: 25280541
[20]	张凯. 溶血磷脂酸酰基转移酶2和5调控甘蓝型油菜种子油脂合成的功能研究[D]. 武汉: 华中科技大学, 2021.doi:10.27157/d.cnki.ghzku.2021.002402.
	Zhang K. Study on the function of lysophosphatidic acid acyltransferase 2 and 5 in regulating oil synthesis of Brassica napus seeds[D]. Wuhan: Huazhong University of Science and Technology, 2021.
[21]	Zhang K, Nie L L, Cheng Q Q, Yin Y T, Chen K, Qi F Y, Zou D S, Liu H H, Zhao W G, Wang B S, Li M T. Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed(Brassica napus L.)using CRISPR-Cas9 system[J]. Biotechnology for Biofuels, 2019, 12(1):225.doi:10.1186/s13068-019-1567-8.
[22]	肖旦望, 刘聪, 胡学芳, 陈社员, 官春云, 熊兴华. 甘蓝型油菜LPAT4基因的克隆与表达[J]. 作物学报, 2014, 40(10):1748-1755.doi:10.3724/SP.J.1006.2014.01748.
	Xiao D W, Liu C, Hu X F, Chen S Y, Guan C Y, Xiong X H. Cloning and expression of LPAT4 genes from Brassica napus[J]. Acta Agronomica Sinica, 2014, 40(10):1748-1755.
[23]	汪慧慧, 刘少锋, 岳宁燕, 熊兴华. 甘蓝型油菜BnaLPAT2基因克隆与生物信息学分析及载体构建[J]. 基因组学与应用生物学, 2019, 38(4):1666-1673.doi:10.13417/j.gab.038.001666.
	Wang H H, Liu S F, Yue N Y, Xiong X H. Cloning and bioinformatics analysis of BnaLPAT2 genes in Brassica napus and construction of their plant expression vector[J]. Genomics and Applied Biology, 2019, 38(4):1666-1673.
[24]	邢蔓, 洪波, 张泽荣, 张曦予, 陈拓, 吴宁柔, 官梅, 邬贤梦, 官春云, 熊兴华. Napin启动子特异性表达油菜BnGPAT9基因[J]. 华北农学报, 2020, 35(4):1-7.doi:10.7668/hbnxb.20190708.
	Xing M, Hong B, Zhang Z R, Zhang X Y, Chen T, Wu N R, Guan M, Wu X M, Guan C Y, Xiong X H. Seed-specific expression of BnGPAT9 gene controlled by napin promoter in Arabidopsis thaliana[J]. Acta Agriculturae Boreali-Sinica, 2020, 35(4):1-7.
[25]	常涛, 李纲, 官梅, 张振乾. 不同含油量油菜籽代谢物差异分析[J]. 分子植物育种, 2021, 19(16):5496-5502.doi:10.13271/j.mpb.019.005496.
	Chang T, Li G, Guan M, Zhang Z Q. Analysis on the difference of metabolites in rapeseed with different oil content[J]. Molecular Plant Breeding, 2021, 19(16):5496-5502.
[26]	Muñoz C F, Weusthuis R A, D'Adamo S, Wijffels R H. Effect of single and combined expression of lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase,and diacylglycerol acyltransferase on lipid accumulation and composition in Neochloris oleoabundans[J]. Frontiers in Plant Science, 2019, 10:1573.doi:10.3389/fpls.2019.01573.
[27]	张军平, 江木兰, 龚阳敏, 万霞, 梁焯, 黄凤洪. 三角褐指藻LPAAT基因在酵母中表达对脂肪酸的影响[J]. 中国油料作物学报, 2012, 34(5):483-488.
	Zhang J P, Jiang M L, Gong Y M, Wan X, Liang Z, Huang F H. Influence of expression of Phaeodactylum tricornutum LPAAT gene in yeast on oil content and fatty acid composition of TAG[J]. Chinese Journal of Oil Crop Sciences, 2012, 34(5):483-488.
[28]	Barroga N A M, Nakamura Y. LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2(LPAT2)is required for de novo glycerolipid biosynthesis,growth,and development in vegetative and reproductive tissues of Arabidopsis[J]. The Plant Journal, 2022, 112(3):709-721.doi:10.1111/tpj.15974. pmid: 36226675
[29]	Yin Y T, Raboanatahiry N, Chen K, Chen X F, Tian T, Jia J, He H S, He J J, Guo Z Y, Yu L J, Li M T. Class A lysophosphatidic acid acyltransferase 2 from Camelina sativa promotes very long-chain fatty acids accumulation in phospholipid and triacylglycerol[J]. The Plant Journal, 2022, 112(5):1141-1158.doi:10.1111/tpj.15999.
[30]	Shaikh A A, Alamin A, Jia C X, Gong W, Deng X J, Shen Q W, Hong Y Y. The examination of the role of rice lysophosphatidic acid acyltransferase 2 in response to salt and drought stresses[J]. International Journal of Molecular Sciences, 2022, 23(17):9796.doi:10.3390/ijms23179796.
[31]	Angkawijaya A E, Nguyen V C, Nakamura Y. Enhanced root growth in phosphate-starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of lysophosphatidic acid acyltransferase 2(LPAT2)[J]. Plant,Cell & Environment, 2017, 40(9):1807-1818.doi:10.1111/pce.12988.
[32]	Shockey J, Regmi A, Cotton K, Adhikari N, Browse J, Bates P D. Identification of Arabidopsis GPAT9(At5g60620)as an essential gene involved in triacylglycerol biosynthesis[J]. Plant Physiology, 2015, 170(1):163-179.doi:10.1104/pp.15.01563.
[33]	Misra A, Khan K, Niranjan A, Kumar V, Sane V A. Heterologous expression of two GPATs from Jatropha curcas alters seed oil levels in transgenic Arabidopsis thaliana[J]. Plant Science, 2017, 263:79-88.doi:10.1016/j.plantsci.2017.07.003.
[34]	Rao S S, Hildebrand D. Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene[J]. Lipids, 2009, 44(10):945-951.doi:10.1007/s11745-009-3337-z.

引物名称 Primer name	正向引物序列(F) Forward primer sequence(F)	反向引物序列(R) Reverse primer sequence(R)
LPAT2-35S	GAAGGTGGCTCCTACAAATGC	GAGAACCACATTGACCAGCCT
LPAT2-Np	TCACTCCCTTCAAACACCTAA	TAGTCTTAGCATCGTTGGGGG
BnaLPAT2-A07	TTGGGGATTCTCTTCTTCATT	CTCTGAGCGAGAATCCATCCA

引物名称 Primer name	正向引物序列(F) Forward primer sequence(F)	反向引物序列(R) Reverse primer sequence(R)
LPAT2-35S	GAAGGTGGCTCCTACAAATGC	GAGAACCACATTGACCAGCCT
LPAT2-Np	TCACTCCCTTCAAACACCTAA	TAGTCTTAGCATCGTTGGGGG
BnaLPAT2-A07	TTGGGGATTCTCTTCTTCATT	CTCTGAGCGAGAATCCATCCA

转化子 Transformants	软脂酸(C16:0) Palmitic acid	硬脂酸(C18:0) Stearic acid	油酸(C18:1) Oleic acid	亚油酸(C18:2) Linoleic acid	亚麻酸(C18:3) Linolenic acid
WT	4.88±0.12b	1.92±0.24a	59.49±0.07a	24.22±0.19a	9.49±0.11b
35S#1	5.06±0.07a	1.75±0.09b	57.66±0.11b	22.98±0.31a	12.55±0.15a
35S#2	5.12±0.13a	1.91±0.20ab	58.26±0.13b	22.53±0.27b	12.18±0.22a
35S#3	4.98±0.18ab	1.77±0.08b	58.68±0.21b	21.44±0.12b	13.13±0.17a

转化子 Transformants	软脂酸(C16:0) Palmitic acid	硬脂酸(C18:0) Stearic acid	油酸(C18:1) Oleic acid	亚油酸(C18:2) Linoleic acid	亚麻酸(C18:3) Linolenic acid
WT	4.88±0.12b	1.92±0.24a	59.49±0.07a	24.22±0.19a	9.49±0.11b
35S#1	5.06±0.07a	1.75±0.09b	57.66±0.11b	22.98±0.31a	12.55±0.15a
35S#2	5.12±0.13a	1.91±0.20ab	58.26±0.13b	22.53±0.27b	12.18±0.22a
35S#3	4.98±0.18ab	1.77±0.08b	58.68±0.21b	21.44±0.12b	13.13±0.17a

转化子 Transformants	软脂酸(C16:0) Palmitic acid	硬脂酸(C18:0) Stearic acid	油酸(C18:1) Oleic acid	亚油酸(C18:2) Linoleic acid	亚麻酸(C18:3) Linolenic acid
WT	4.88±0.12b	1.92±0.24a	59.49±0.07a	24.22±0.19b	9.49±0.11b
Napin#1	4.75±0.11b	1.48±0.22b	57.26±0.22b	25.99±0.36a	10.52±0.29a
Napin#2	4.84±0.16b	1.66±0.08b	55.85±0.33b	26.14±0.45a	11.65±0.18a
Napin#3	5.01±0.23a	1.43±0.14b	57.03±0.41b	25.83±0.31a	10.71±0.37a

Please choose a citation manager

Content to export