甘蓝型油菜<i>LPAT2-A07</i>基因调控种子含油量和脂肪酸组成

doi:10.7668/hbnxb.20194849

摘要/Abstract

摘要：

为探究甘蓝型油菜溶血磷脂酸酰基转移酶2(LPAT2)基因的功能,从甘蓝型油菜中PCR 同源克隆了BnaLPAT2的一个拷贝(A07),将其构建过表达载体p35S∷BnaLPAT2-A07和种子特异表达载体pNapin∷BnaLPAT2-A07,利用农杆菌介导法遗传转化甘蓝型油菜中双6号,通过转化植株的PCR检测,分别获得15株和11株转基因阳性植株。经实时荧光定量(Real-time Quantitative PCR,qRT-PCR)检测,过表达T₃油菜多数组织中BnaLPAT2-A07基因的表达量较野生型均有显著提升;而种子特异性表达的T₃油菜各组织中BnaLPAT2-A07基因都集中在种子的发育与成熟期超强表达。索氏抽提法检测到由35S启动子或Napin启动子驱动的BnaLPAT2-A07转基因油菜种子中的含油量较野生型分别提升1.39,2.36百分点。气相色谱法检测转基因油菜的脂肪酸组分结果显示,亚麻酸的含量较野生型分别提升3.13,1.47百分点。研究结果表明,BnaLPAT2-A07基因具有促进甘蓝型油菜种子油脂合成的功能,但BnaLPAT2-A07对亚麻酸特异选择性功能还需进一步验证。

关键词: 甘蓝型油菜, 过表达, BnaLPAT2-A07, 脂肪酸分析, 含油量

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

秦艺, 刘勇, 熊兴华. 甘蓝型油菜LPAT2-A07基因调控种子含油量和脂肪酸组成[J]. 华北农学报, 2025, 40(1): 45-52. doi: 10.7668/hbnxb.20194849.

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.

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

图/表 7

表1 引物名称与序列

Tab.1 List of primers name and sequence

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

图1 转基因油菜PCR检测 A.过表达p35S∷BnaLPAT2-A07 PCR检测;B.种子特异表达pNapin∷BnaLPAT2-A07 PCR检测。M.DL2000 DNA Marker;P.阳性质粒;W.野生型;T.阳性株。

Fig.1 PCR detection of transgenic rapeseeds A.Overexpression of p35S∷BnaLPAT2-A07 PCR detection;B.Seed-specific expression of pNapin∷BnaLPAT2-A07 PCR detection.M.DL2000 DNA Marker;P.Positive plasmid;W.Wild-type;T.Positive plant.

表2 转基因油菜鉴定

Tab.2 Identification of transgenic rapeseeds

载体 The vector	总株数 The total number	PCR阳性株数 Number of PCR positive plants	阳性率/% Positive rate
p35S∷BnaLPAT2-A07	18	15	83.33
pNapin∷BnaLPAT2-A07	16	11	68.75

图2 qRT-PCR检测转基因油菜BnaLPAT2-A07的表达不同小写字母表示BnaLPAT2-A07在转基因株系与野生型中表达的差异显著(P<0.05)。图3同。

Fig.2 qRT-PCR detection of the expression of BnaLPAT2-A07 in transgenic rapeseed Lowercase letters denote statistically significant differences in BnaLPAT2-A07 expression levels between the transgenic rapeseeds and the wild-type controls(P<0.05).The same as Fig.3.

表3 p35S∷BnaLPAT2-A07过表达油菜种子脂肪酸组分

Tab.3 Fatty acid components in p35S∷BnaLPAT2-A07 overexpressed rapeseeds%

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

表4 pNapin∷BnaLPAT2-A07过表达油菜种子脂肪酸组分

Tab.4 Fatty acid components in pNapin∷BnaLPAT2-A07 overexpressed rapeseeds%

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

图3 转基因油菜种子含油量检测

Fig.3 Detection of oil content in transgenic rapeseeds

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甘蓝型油菜LPAT2-A07基因调控种子含油量和脂肪酸组成

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