山杏油体蛋白基因PsOLE4克隆及其调控油脂累积功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0251

摘要/Abstract

摘要：

油体蛋白OLE在植物油脂合成累积中具有重要调控作用。依据山杏（Prunus sibirica）不同发育阶段种子的mRNA转录组测序数据，注释获得5个具有完整开放阅读框和典型保守结构域oleosin的油体蛋白OLE家族基因，通过差异转录谱分析及RT-qPCR检测，确定与山杏种子发育及油脂累积密切相关的高表达PsOLE4基因为研究对象，克隆该基因并进行生物信息学分析、组织特异表达检测、亚细胞定位分析、遗传转化拟南芥及其种子油脂含量和脂肪酸组分测定等研究。结果显示，PsOLE4基因全长序列为378 bp，可编码含125个氨基酸且分子量为13 kD的蛋白。该蛋白为疏水性无信号肽的非分泌蛋白，含有18个磷酸化位点、2个跨膜结构域和1个高度保守的脯氨酸结（PX₅SP₃P）结构域，定位于细胞膜上。PsOLE4基因在山杏种子中的表达量显著高于茎、叶及果实，而且PsOLE4基因表达可有效促进转基因拟南芥种子的脂肪酸含量提高与油脂累积，表明山杏PsOLE4基因具有种子表达特异性，对油脂累积具有重要调控作用。研究结果为后续开展山杏种子PsOLE4功能鉴定及应用奠定基础。

关键词: 油体蛋白, 油脂, 山杏种子, PsOLE4基因克隆, 组织表达特性, 亚细胞定位, 遗传转化, 功能分析

Abstract:

Oleosin（OLE）plays an important role in regulating synthesis and accumulation of plant lipids. Based on our recent mRNA transcriptomics sequencing data for the seeds of Prunus sibirica from different developing stages，a total of 5 OLE family genes with complete open reading frame and typical conserved oleosin domain were screened by functional annotation，and importantly，RT-qPCR and differential transcription profile were used to determine the PsOLE4 with abundant transcript closely related to development and oil accumulation of P. sibirica seeds as research objects. Hence，the PsOLE4 gene was cloned from P. sibirica seeds，and then the analyses of bioinformatics，tissue-specific expression，subcellular localization and ectopic expression in Arabidopsis thaliana as well as oil content and fatty acid composition of transgenic seeds were conducted. The results showed that the full-length cDNA of PsOLE4 contained an open reading frame of 378 bp in length，encoding a protein of 125 amino acid residues with a molecular mass of 13 kD. The PsOLE4 protein，without signal peptide，was a non-secretory and hydrophobic protein containing 18 phosphorylation sites，2 transmembrane domains and a highly conserved proline junction domain（PX₅SP₃P），which was mainly localized on cell membrane. The expressions of PsOLE4 was much higher in the seeds of P. sibirica than in the stems，leaves and fruits，and PsOLE4 expression efficiently enhanced the contents of oils and its five predominant fatty acids in A. thaliana seeds，revealing that PsOLE4 with seed-expressed specificity may contribute to the oil accumulation of transgenic A. thaliana seeds. In conclusion，our results could provide important foundation for the further studies of functional identification of PsOLE4 and its application for oil yield improvement.

Key words: oleosin, oil, Prunus sibirica seeds, PsOLE4 gene cloning, tissue specific expression, subcellular localization, genetic transformation, function analysis

党瑗, 李维, 苗向, 修宇, 林善枝. 山杏油体蛋白基因PsOLE4克隆及其调控油脂累积功能分析[J]. 生物技术通报, 2022, 38(11): 151-161.

DANG Yuan, LI Wei, MIAO Xiang, XIU Yu, LIN Shan-zhi. Cloning of Oleosin Gene PsOLE4 from Prunus sibirica and Its Regulatory Function Analysis for Oil Accumulation[J]. Biotechnology Bulletin, 2022, 38(11): 151-161.

图/表 9

表1 实验所用引物及序列

Table 1 Primers and sequences used in the study

引物 Primer	序列 Sequence（5'-3'）	备注 Annotation
PsOLE1-qf	CTTTGACCATCGCCACTCCG	PsOLE1基因荧光定量引物
PsOLE1-qr	CGCCACACCGAATCCACC	PsOLE1基因荧光定量引物
PsOLE2-qf	GAGACGGCGGGCTATTT	PsOLE2基因荧光定量引物
PsOLE2-qr	CAGCATCCTGGGACTTATCTAC	PsOLE2基因荧光定量引物
PsOLE3-qf	GCCTTCCCAGCCAACTTAT	PsOLE3基因荧光定量引物
PsOLE3-qr	TTCTTGACTTCCGGTGGATTC	PsOLE3基因荧光定量引物
PsOLE4-qf	ACGTTAACTGGGACAGTGATG	PsOLE4基因荧光定量引物
PsOLE4-qr	GCTGTCAGGAACACGACTATAC	PsOLE4基因荧光定量引物
PsOLE5-qf	TCTTGGGTCACCAACTACCT	PsOLE5基因荧光定量引物
PsOLE5-qr	GTCCCACAAACTCCACCATATC	PsOLE5基因荧光定量引物
CYP-qf	CAACGGATCTCAGTTCTTCGTCTGC	CYP内参基因荧光定量引物
CYP-qr	GACCCAACCTTCTCGATGTTCTTCA	CYP内参基因荧光定量引物
UBC-qf	GAGACCAGCAATAACCGTGAA	UBC内参基因荧光定量引物
UBC-qr	TCTTGTACTCCGTGGCATCCT	UBC内参基因荧光定量引物
PsOLE4-f1	CTCTTTGAGCAACTAATGACGTACA	PsOLE4克隆引物
PsOLE4-r1	ATTATCCAAACAACCCAACTTACCC	PsOLE4克隆引物
PsOLE4-f2	TGGAGAGAACACGGGGGACTCTAGAATGGCTGATCAATCAAGACACGTC	PsOLE4亚细胞定位引物
PsOLE4-r2	GGTGGCGACCGGTACCCGGGGATCAAAAGGGCAGAAGTACTGCCCATAC	PsOLE4亚细胞定位引物
PsOLE4-f3	GAGAGAACACGGGGGACTCTAGAGATGGCTGATCAATCAAGACACGTC	PsOLE4表达载体引物
PsOLE4-r3	GAACGATCGGGGAAATTCGAGCTTCAAAAAGGGCAGAAGTACTGCCCA	PsOLE4表达载体引物

图1 山杏种子PsOLE蛋白保守结构域及PsOLE基因的动态转录表达水平分析 A：山杏种子PsOLE蛋白保守结构域分析；B：mRNA高通量测序分析PsOLE家族基因表达水平，以log10（FPKM）进行标准化；C：RT-qPCR测定PsOLE家族基因相对表达水平，以log10（相对表达量）进行标准化。图中误差线为标准差

Fig. 1 Analysis of conserved domain for PsOLEs and dynamic transcriptional expression of PsOLEs in P. sibirica seeds A：Analysis of conserved domain for PsOLE proteins in P. sibirica seeds. B：Expressions level of PsOLE family genes by mRNA high-throughput sequencing，the values were normalized as log10（FPKM）. C：Determination of relative expressions of PsOLE family genes by RT-qPCR，the values were normalized as log10（relative expression）. The bar in the figure refers to the standard deviation

图2 山杏种子PsOLE4基因的扩增电泳图谱 M：DNA marker 2000；1：PsOLE4的基因片段

Fig. 2 Amplification electrophoretic map of the PsOLE4 gene from P. sibirica seeds M：2000 DNA marker. 1：PsOLE4 gene fragment

图3 山杏种子PsOLE4蛋白生物信息学分析 A：山杏种子PsOLE4蛋白磷酸化位点分析；B：PsOLE4蛋白亲/疏水性分析；C：PsOLE4蛋白信号肽分析；D：PsOLE4蛋白跨膜结构分析；E：PsOLE4蛋白二级结构预测；F：PsOLE4蛋白三级结构预测

Fig. 3 Bioinformatics analysis for PSOLE4 protein from P. sibirica seeds A：Analysis of phosphorylation site for PsOLE4 protein from P. sibirica seeds. B：Analysis of hydrophilicity/hydrophobicity for PsOLE4. C：Analysis of signal peptide for PsOLE4. D：Analysis of transmembrane region for PsOLE4. E：Prediction of secondary structure for PsOLE4. F：Prediction of tertiary structure for PsOLE4

图4 山杏种子PsOLE4蛋白的多序列比对山杏（Prunus sibirica），甜樱桃（Prunus mume），梅花（Prunus mume），胡杨（Populus euphratica），毛果杨（Populus trichocarpa），野毛豆（Glycine soja），野茶树（Camellia sinensis），水稻（Oryza sativa），雀麦（Bromus secalinus），巴坦杏（Prunus dulcis），碧桃（Prunus persica），苹果（Malus domestica），白梨（Pyrus × bretschneideri），草莓（Fragaria vesca），月季（Rosa chinensis），日本樱花（Prunus yedoensis），大豆（Glycine max），拟南芥（Arabidopsis thaliana），荠菜（Capsella rubella），油菜（Brassica napus），麻疯树（Jatropha curcas），巨桉（Eucalyptus grandis），油棕（Elaeis guineensis），咖啡树（Coffea eugenioides），蓖麻（Ricinus communis），芝麻（Sesamum indicum）

Fig. 4 Multiple sequence alignment of PsOLE4 protein from P. sibirica seeds

图5 山杏种子PsOLE4蛋白的系统进化树分析

Fig. 5 Analysis of phylogenetic tree for PsOLE4 protein from P. sibirica seeds

图6 山杏PsOLE4基因在不同组织中的相对表达量分析以叶中PsOLE4基因的最低表达量为对照，标准化校正为1

Fig. 6 Analysis of relative expression for PsOLE4 gene in the different tissues of P. sibirica The lowest expression of PsOLE4 gene in the leaves was taken as control，and standardized calibration is 1

图7 山杏种子PsOLE4蛋白的亚细胞定位 Dark为荧光照片；Bright为明场照片；Merged为合并照片

Fig. 7 Subcellular localization of PsOLE4 from P. sibirica seeds Dark is defined as dark-field fluorescence image. Bright is defined as bright-field image. Merged is defined as the merged image in both bright and dark fields

图8 转PsOLE4基因拟南芥的生长状态及其种子脂肪酸含量与组分分析 A：转基因拟南芥PCR鉴定，M：DNA marker，1：空载体植株（empty）PCR产物，2-4：转PsOLE4基因植株（35S∷PsOLE4 #1、35S∷PsOLE4 #2和35S∷PsOLE4 #3）PCR产物；B：野生型（WT）、空载体和过表达PsOLE4基因拟南芥生长状况，比例尺=5 cm；C：野生型、空载体和过表达PsOLE4基因拟南芥种子脂肪酸含量测定；D：拟南芥种子脂肪酸组分分析。星号（*）表示与野生型拟南芥差异显著P < 0.05。C16：0：棕榈酸；C18：0：硬脂酸；C18：1：油酸；C18：2：亚油酸；C18：3：亚麻酸；C20：0：花生酸；C20：1：花生油酸

Fig. 8 Analyses for growth status in PsOLE- transgenic A. thaliana and fatty acid content and composition of their seeds A：PCR identification for transgenic A. thaliana，M：DNA marker，1：PCR product from empty vector transgenic plant（empty），2-4：PCR product from PsOLE4 transgenic plants（35S∷PsOLE4 #1、35S∷PsOLE4 #2和35S∷PsOLE4 #3）. B：Growth status of WT，empty and PsOLE4 overexpressing A. thaliana lines，scale bar=5 cm. C：Fatty acid contents in the seeds of WT，empty and PsOLE4 overexpressing transgenic A. thaliana lines. D：Fatty acid composition in seeds of A. thaliana. The asterisks（*）above the columns indicate significant differences at P<0.05. C16∶0：Palmitic acid. C18：0：Stearic acid. C18：1：Oleic acid. C18：2：Linoleic acid. C18：3：Linolenic acid. C20：0：Arachidic acid. C20：1：Eicosenoic acid

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