番木瓜CpWRI3参与调控果实成熟过程中脂肪酸合成

doi:10.16420/j.issn.0513-353x.2022-0590

摘要/Abstract

摘要：

以番木瓜（Carica papaya L.）果实为研究材料，通过前期构建的果实成熟数字基因表达谱，克隆得到1个编码WRI家族调控因子的基因，命名为CpWRI3。氨基酸序列比对和进化分析发现CpWRI3与拟南芥AtWRI3/4同源性较高。转录因子活性分析表明，CpWRI3具有一定的转录激活活性。实时荧光定量PCR分析表明，CpWRI3的表达水平随着果实成熟不断升高，采后8 d达到峰值，表明其在果实成熟的后期发挥重要作用。在果实中瞬时过量表达CpWRI3促进了月桂酸、棕榈油酸和亚油酸含量的提升。体外凝胶阻滞试验（EMSA）证实CpWRI3能与下游靶基因CpKAS1和CpACC1启动子上AW-box元件的序列特异结合。利用荧光素酶系统进一步证明CpWRI3可以在植物体内诱导CpKAS1和CpACC1启动子的表达。上述结果表明CpWRI3能参与调控月桂酸、棕榈油酸和亚油酸等脂肪酸合成。

关键词: 番木瓜, 果实, 成熟, 脂肪酸, 转录调控, WRI家族

Abstract:

A WRI family regulator gene named CpWRI3 was cloned from papaya fruit. Amino acid sequence alignment and evolutionary analyses showed that CpWRI3 has high homology with AtWRI3/4. Transcriptional factor activity analysis showed that CpWRI3 has transcriptional activate function. The expression level of CpWRI3 increased during the fruit ripening process，peaked at eight days after harvested，indicating that CpWRI3 plays an important role in the late stage of fruit ripening. In order to explore the role of CpWRI3 in regulating fatty acid biosynthesis，transient overexpression of CpWRI3 was performed. The data showed that overexpression of CpWRI3 significantly induced the contents of lauric acid，palmitoleic acid and linoleic acid in papaya fruit. In vitro Electrophoretic Mobility Shift Assay（EMSA）confirmed that CpWRI3 could specifically bind to AW-box elements on promoters of downstream target genes CpKAS1 and CpACC1. Using luciferase system，CpWRI3 can induce the promoter activities of CpKAS1 and CpACC1 genes in plants. These results revealed that CpWRI3 may induce the biosynthesis of lauric acid，palmitoleic acid and linoleic acid.

Key words: papaya, fruit, ripening, fatty acid, transcription regulation, WRI family

莫雨杏, 周艳, 林润婷, 多泳星, 张涛, 刘锴栋. 番木瓜CpWRI3参与调控果实成熟过程中脂肪酸合成[J]. 园艺学报, 2023, 50(8): 1637-1648.

MO Yuxing, ZHOU Yan, LIN Runting, DUO Yongxing, ZHANG Tao, LIU Kaidong. Papaya CpWRI3 is Involved in the Regulation of Fatty Acid Accumulation During Fruit Postharvest[J]. Acta Horticulturae Sinica, 2023, 50(8): 1637-1648.

图/表 11

表1 引物序列

Table 1 Primer sequences

引物用途 Purpose	引物名称 Primier name	引物序列（5′-3′） Sequence
CpWRI3基因扩增 Amplification of CpWRI3 gene	CpWRI3-F	CAATGCACAGAATGGTGCTGG
CpWRI3基因扩增 Amplification of CpWRI3 gene	CpWRI3-R	TTGAAACATGGTCGGCACGAG
荧光定量检测引物 Real-time PCR of CpWRI3	CpWRI3-RT-F	GCACATACGCAACGCAAGAA
荧光定量检测引物 Real-time PCR of CpWRI3	CpWRI3-RT-R	TTCGGGATTAGGGTTTGGCG
荧光定量内参引物 Real-time PCR of CpActin	CpActin-RT-F	CTGTGCATGATGACTGCAAG
荧光定量内参引物 Real-time PCR of CpActin	CpActin-RT-R	TCATCTTGCTTCTCACCTTG

表2 荧光定量检测引物序列

Table 2 Primer sequences of Real-time PCR

目的基因 Gene	引物序列（5′-3′） Sequence
CpWRI3	F：TGCTGGAACGAATCGCAGAA;R：TCGTGCAACGCCTCTGTATT
CpKAS1	F：TGCGCATGCAACTTCAACTC;R：AACGTTCACCTCATGTCGCT
CpACP2	F：AAGTATGGTGATCGGGTGGC;R：TCCTGATCCCCTCACCACAT
CpBCCP2	F：GCGTTGGCTCAACCACAATC;R：CGACCTTCACAAAGGGTGGT
CpACC1	F：TAGCTGTGCGTGTGACAAGT;R：TTGGTTCGGATTTCTCCCCG

图1 CpWRI3基因PCR产物电泳图

Fig. 1 The PCR product of CpWRI3 gene Marker：DL5 000.

图2 CpWRI3蛋白3D结构图

Fig. 2 3D structure of CpWRI3 protein

图3 番木瓜CpWRI3与拟南芥（At）WRI氨基酸多序列比对（A）及与其他物种WRI蛋白同源进化分析（B） B中分支上的数据代表自展值。

Fig. 3 Alignment of the predicted CpWRI3 protein between papaya and Arabidopsis thaliana（A）and the phylogenetic tree of CpWRI3 protein and WRI proteins from other species（B） The data of B on the branch represents the bootstrap value.

图4 CpWRI3转录因子活性分析

Fig. 4 Transcriptional activity of CpWRI3 Student’s t-test，*** P < 0.001.

图5 番木瓜果实成熟过程中CpWRI3的表达量变化

Fig. 5 Expression analysis of CpWRI3 gene during the fruit ripening process

图6 番木瓜CpWRI3瞬时过表达（OE）和对照果实中CpWRI3及其相关基因表达变化

Fig. 6 The expression change of CpWRI3 and relaive genesin CpWRI3 transient overexpressing（OE）and the control fruit Student’s t-test. **P < 0.01;***P < 0.001.

图7 番木瓜CpWRI3瞬时过表达（OE）和对照果实中月桂酸、棕榈油酸、亚油酸含量变化

Fig. 7 The changes of content of lauric acid，palmitoleic acid，and linoleic acidin CpWRI3 transient overexpressing（OE）and the control fruit Student’s t-test. **P < 0.01;***P < 0.001.

图8 CpWRI3蛋白与CpKAS1和CpACC1启动子AW-box元件特异结合

Fig. 8 CpWRI3 protein was specifically binding to the AW-box in promoter of CpKAS1 and CpACC1 genes

图9 瞬时表达分析中用到的载体（A）和相对LUC/REN值（B）

Fig. 9 Vectors used in transient expression analysis（A）and relative LUC/REN ratio（B）

参考文献 30

[1]	Baud S, Mendoza M S, To A, Harscoet E, Lepiniec L, Dubreucq B. 2007. WRINKLED 1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis. The Plant Journal, 50 (5):825-838. doi: 10.1111/tpj.2007.50.issue-5 URL
[2]	Behera J R, Rahman M M, Bhatia S, Shockey J, Kilaru A. 2021. Functional and predictive structural characterization of WRINKLED2,a unique oil biosynthesis regulator in Avocado. Frontiers in Plant Science, 12:648494. doi: 10.3389/fpls.2021.648494 URL
[3]	Cernac A, Benning C. 2004. WRINKLED 1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. The Plant Journal, 40 (4):575-585.
[4]	Chen B, Zhang G, Li P, Yang J, Guo L, Benning C, Wang X, Zhao J. 2020. Multiple GmWRI1s are redundantly involved in seed filling and nodulation by regulating plastidic glycolysis,lipid biosynthesis and hormone signalling in soybean(Glycine max). Plant Biotechnology Journal, 18 (1):155-171. doi: 10.1111/pbi.v18.1 URL
[5]	Chen Yongping, Gao Feng, Shen Yanhong, Zhao Wanwan, Chen Guixin, Wang Ping. 2019. The analysis of ERFs related to fruit ripening in papaya. Acta Horticulturae Sinica, 46 (2):252-264. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-0396
	陈永萍, 高峰, 申艳红, 赵湾湾, 陈桂信, 王平. 2019. 番木瓜ERF家族与果实成熟相关成员的分析. 园艺学报, 46 (2):252-264. doi: 10.16420/j.issn.0513-353x.2018-0396
[6]	Deshpande A B, Chidley H G, Oak P S, Pujari K H, Giri A P, Gupta V S. 2016. Data on changes in the fatty acid composition during fruit development and ripening of three mango cultivars(Alphonso,Pairi and Kent)varying in lactone content. Data in Brief, 9:480-491. pmid: 27722190
[7]	Elahi N, Duncan R W, Stasolla C. 2015. Decreased seed oil production in FUSCA3 Brassica napus mutant plants. Plant Physiology and Biochemistry, 96:222-230. doi: 10.1016/j.plaphy.2015.08.002 URL
[8]	Hanano A, Almousally I, Shaban M, Murphy D J. 2018. Arabidopsis plants exposed to dioxin result in a WRINKLED seed phenotype due to 20S proteasomal degradation of WRI1. Journal of Experimental Botany, 69 (7):1781-1794. doi: 10.1093/jxb/ery027 URL
[9]	Huang Jingyi, Xie Jingxian, Ke Desen. 2019. Study on effect of maturing mode on nutritional composition of papaya by chemical analysis. Guangzhou Chemical Industry, 47 (14):118-120. (in Chinese)
	黄靖怡, 谢靖娴, 柯德森. 2019. 化学分析法研究成熟方式对番木瓜营养成份的影响. 广州化工, 47 (14):118-120.
[10]	Jha S N, Jaiswal P, Narsaiah K, Kaur P P, Singh A K, Kumar R. 2013. Textural properties of mango cultivars during ripening. Journal of Food Science and Technology, 50 (6):1047-1057. doi: 10.1007/s13197-011-0431-z pmid: 24426016
[11]	Jiang B, Ou S Y, Xu L, Mai W Y, Ye M J, Gu H P, Zhang T, Yuan C C, Shen C J, Wang J X, Liu K D. 2019. Comparative proteomic analysis provides novel insights into the regulation mechanism underlying papaya(Carica papaya L.)exocarp during fruit ripening process. BMC Plant Biology, 19 (1):238. doi: 10.1186/s12870-019-1845-4 pmid: 31170911
[12]	Jiang Bian, Pan Jiaqi, Ye Liuqing, Lu Lishi, Yuan Changchun, Liu Kaidong. 2018. Transcriptome data assembly and gene function annotation of Carica papaya fruits. Molecular Plant Breeding, 16 (21):6945-6954. (in Chinese)
	蒋边, 潘嘉琪, 叶柳清, 陆丽施, 袁长春, 刘锴栋. 2018. 番木瓜果实转录组数据组装及基因功能注释. 分子植物育种, 16 (21):6945-6954.
[13]	Kong Q, Ma W. 2018. WRINKLED 1 transcription factor:how much do we know about its regulatory mechanism? Plant Science, 272:153-156. doi: S0168-9452(18)30126-2 pmid: 29807586
[14]	Li Q, Shao J, Tang S, Shen Q, Wang T, Chen W, Hong Y. 2015. Wrinkled 1 accelerates flowering and regulates lipid homeostasis between oil accumulation and membrane lipid anabolism in Brassica napus. Frontiers in Plant Science, 6:1015.
[15]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[16]	Ma Qian, Li Jingbin, Ruan Chengjiang, Li Rongrong. 2016. Bioinformatics analysis of WRI1 gene in Hippophae rhamnoides. Hubei Agricultural Sciences, 55 (22):5872-5975,5981. (in Chinese)
	马倩, 李景滨, 阮成江, 李荣荣. 2016. 沙棘WRI1转录因子基因的生物信息学分析. 湖北农业科学, 55 (22):5872-5975,5981.
[17]	Maeo K, Tokuda T, Ayame A, Mitsui N, Kawai T, Tsukagoshi H, Ishiguro S, Nakamura K. 2009. An AP2-type transcription factor,WRINKLED1,of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis. The Plant Journal, 60 (3):476-487. doi: 10.1111/tpj.2009.60.issue-3 URL
[18]	Mindrebo J T, Patel A, Kim W E, Davis T D, Chen A, Bartholow T G, La Clair J J, McCammon J A, Noel J P, Burkart M D. 2020. Gating mechanism of elongating beta-ketoacyl-ACP synthases. Nature Communication, 11 (1):1727. doi: 10.1038/s41467-020-15455-x
[19]	Mizojiri R, Tomita D, Sasaki M, Satoh Y, Yamamoto Y, Sumi H, Maezaki H. 2021. Design and synthesis of a monocyclic derivative as a selective ACC 1 inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors. Bioorganic & Medicinal Chemistry, 35:116056. doi: 10.1016/j.bmc.2021.116056 URL
[20]	Saini R K, Zamany A J, Keum Y S. 2017. Ripening improves the content of carotenoid,alpha-tocopherol,and polyunsaturated fatty acids in tomato(Solanum lycopersicum L.)fruits. 3 Biotech, 7 (1):43.
[21]	Sun Jinbo, Shi Suhua, Yang Li, Li Fengli, Wang Xingjun, Zhao Shuzhen. 2020. Genome-wide analysis of WRI1gene family and their expression profiles in peanut. Journal of Peanut Science, 49 (1):9-18. (in Chinese)
	孙金波, 石素华, 杨利, 李凤丽, 王兴军, 赵术珍. 2020. 花生WRI1基因家族的全基因组与表达谱分析. 花生学报, 49 (1):9-18.
[22]	Tian Xuebing, Weng Shengyu, Dai Zhihui, Wang Jianqing, Ni Sui. 2016. Nutrient analysis of three varieties of papaya(Carica papaya L.)under protected cultivation. Chinese Wild Plant Resources, 35 (5):14-19. (in Chinese)
	田雪冰, 翁生余, 戴智慧, 王建青, 倪穗. 2016. 设施栽培下3个品种番木瓜的营养成分分析. 中国野生植物资源, 35 (5):14-19.
[23]	To A, Joubes J, Barthole G, Lecureuil A, Scagnelli A, Jasinski S, Lepiniec L, Baud S. 2012. WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis. The Plant Cell, 24 (12):5007-5023. doi: 10.1105/tpc.112.106120 URL
[24]	Wang J J, Liu H R, Gao J, Huang Y J, Zhang B, Chen K S. 2016. Two omega-3 FADs are associated with peach fruit volatile formation. International Journal of Molecular Sciences, 17 (4):464. doi: 10.3390/ijms17040464 URL
[25]	Xu Y X, Liu J J, Zang N N, Yin Z P, Wang A D. 2022. Effects of calcium application on apple fruit softening during storage revealed by proteomics and phosphoproteomics. Horticultural Plant Journal, 8 (4):408-422. doi: 10.1016/j.hpj.2022.03.002 URL
[26]	Yang Xianyou, Huang Youjun, Zhang Tong, Huang Chunying. 2016. Advances on transcriptional activator AtWRI1of Arabidopsis. Biotechnology Bulletin, 32 (6):13-18. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2016.06.004
	杨先友, 黄有军, 张通, 黄春颖. 2016. 拟南芥转录激活因子AtWRI1研究进展. 生物技术通报, 32 (6):13-18. doi: 10.13560/j.cnki.biotech.bull.1985.2016.06.004
[27]	Yang Y, Munz J, Cass C, Zienkiewicz A, Kong Q, Ma W, Sanjaya, Sedbrook J, Benning C. 2015. Ectopic expression of WRINKLED 1 affects fatty acid homeostasis in Brachypodium distachyon vegetative tissues. Plant Physiology, 169 (3):1836-1847. doi: 10.1104/pp.15.01236 pmid: 26419778
[28]	Zhang Q, Sun R, Zheng Y, Yuan Y, Li D. 2019. Isolation and characterization of the EgWRI1promoter from oil palm(Elaeis guineensis Jacq.)and its response to environmental stress and ethylene. PLoS ONE, 14 (12):e0225115. doi: 10.1371/journal.pone.0225115 URL
[29]	Zhang T, Li W J, Xie R X, Xu L, Zhou Y, Li H L, Yuan C C, Zheng X L, Xiao L T, Liu K D. 2020. CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and regulate fruit ripening in papaya. The Plant Journal, 103:1318-1337. doi: 10.1111/tpj.14803 pmid: 32391615
[30]	Zhou Chenping, Yang Min, Guo Jinju, Kuang Ruibin, Yang Hu, Huang Bingxiong, Wei Yuerong. 2022. Dynamic changes in DNA methylome and transcriptome patterns during papaya fruit ripening. Acta Horticulturae Sinica, 49 (3):519-532. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-1046
	周陈平, 杨敏, 郭金菊, 邝瑞彬, 杨护, 黄炳雄, 魏岳荣. 2022. 番木瓜成熟过程中全基因组DNA甲基化和转录组变化分析. 园艺学报, 49 (3):519-532. doi: 10.16420/j.issn.0513-353x.2020-1046