甜瓜果实成熟相关基因家族的全基因组鉴定及分析
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摘要
为获得与甜瓜果实成熟相关的基因,选择与乙烯生物合成及信号转导相关的4个基因家族HB(Homeobox)、RIN(Ripening inhibitor)、ETR(Ethylene receptor)、CTR(Constitutive triple reaction),对甜瓜全基因组鉴定,分别获得CmHB家族成员17个、CmRIN家族成员21个、CmETR家族成员3个和CmCTR家族成员20个,通过序列比对和基序分析验证了家族成员鉴定的可靠性。利用转录组测序方法,分析4个基因家族各成员在甜瓜品种河套蜜瓜原种和转反义CmACO1基因的河套蜜瓜品系M9生长期和成熟期果实中的表达量,发现河套蜜瓜原种中13个基因在2个发育时期果实中的表达量存在显著性差异,其中CmHB3、CmHB11生长期的表达量是成熟期的42,9倍,而CmHB4成熟期的表达量是生长期的27倍,其表达量均呈极显著差异。在M9品系中,12个基因在2个发育时期果实中的表达量存在显著差异,其中CmHB3、CmHB11生长期的表达量是成熟期的6,3倍,而CmHB4成熟期的表达量是生长期的41倍,其表达量均呈极显著差异。在生长期,CmHB3、CmHB11的表达量在2个材料中存在极显著差异,CmRIN14、CmRIN15存在显著差异。而在成熟期,CmHB3的表达量在2个材料中存在极显著差异,CmRIN14、CmRIN15存在显著差异。此外,还发现CmRIN14、CmRIN15在2个材料间的表达模式相反,表明其表达模式受CmACO1表达水平的影响。
To obtain the genes related to fruit maturation of melon, the genome-wide identification of four gene families, HB(Homeobox),RIN(Ripening inhibitor),ETR(Ethylene receptor) and CTR(Constitutive triple reaction) from melon, which were related to ethylene biosynthesis and signal transduction, were carried out in the study. A total of 17 members of CmHB family,21 members of CmRIN family,3 members of CmETR family and 20 members of CmCTRs family were retrieved from the melon genome, which were further verified by sequence alignment and motif analysis. Using transcriptional profiling analysis, the expressions of the four gene family members in wild type Cucumis melo L.cv Hetao and transgenic Cucumis melo L.cv Hetao with antisense CmACO1 gene(M9) were determined during growth period and at ripening stage of fruits. The results showed that 13 genes were differentially expressed between the two developmental stages of fruit in wild type melon, of which the expression amount of CmHB3 and CmHB11 in growth period was, respectively, 42 and 9 times that at ripening stage, but the expression level of CmHB4 at ripening stage was 27 times that in growth period, all reaching to a highly significant level. And 12 genes were also differentially expressed between two developmental stages of fruit in M9 transgenic line, among which the expression amount of CmHB3 and CmHB11 in growth period was, respectively, 6 and 3 times that at ripening stage, while the expression level of CmHB4 at ripening stage was 41 times that in growth period, all the differences being highly significant. During the growth of fruit, the expression levels of CmHB3 and CmHB11 had extremely significant difference, and the expression levels of CmRIN14 and CmRIN15 were significantly different between wild type and M9 fruits. At ripening stage of fruit, the difference in expression level of CmHB3 was highly significant and the differences in expression levels of CmRIN14 and CmRIN15 were significant between two types of melon fruits. In addition, the expression patterns of CmRIN14 and CmRIN15 between the two melon materials presented a opposite trend,indicating that their expression patterns were affected by the expression level of CmACO1.
To obtain the genes related to fruit maturation of melon, the genome-wide identification of four gene families, HB(Homeobox),RIN(Ripening inhibitor),ETR(Ethylene receptor) and CTR(Constitutive triple reaction) from melon, which were related to ethylene biosynthesis and signal transduction, were carried out in the study. A total of 17 members of CmHB family,21 members of CmRIN family,3 members of CmETR family and 20 members of CmCTRs family were retrieved from the melon genome, which were further verified by sequence alignment and motif analysis. Using transcriptional profiling analysis, the expressions of the four gene family members in wild type Cucumis melo L.cv Hetao and transgenic Cucumis melo L.cv Hetao with antisense CmACO1 gene(M9) were determined during growth period and at ripening stage of fruits. The results showed that 13 genes were differentially expressed between the two developmental stages of fruit in wild type melon, of which the expression amount of CmHB3 and CmHB11 in growth period was, respectively, 42 and 9 times that at ripening stage, but the expression level of CmHB4 at ripening stage was 27 times that in growth period, all reaching to a highly significant level. And 12 genes were also differentially expressed between two developmental stages of fruit in M9 transgenic line, among which the expression amount of CmHB3 and CmHB11 in growth period was, respectively, 6 and 3 times that at ripening stage, while the expression level of CmHB4 at ripening stage was 41 times that in growth period, all the differences being highly significant. During the growth of fruit, the expression levels of CmHB3 and CmHB11 had extremely significant difference, and the expression levels of CmRIN14 and CmRIN15 were significantly different between wild type and M9 fruits. At ripening stage of fruit, the difference in expression level of CmHB3 was highly significant and the differences in expression levels of CmRIN14 and CmRIN15 were significant between two types of melon fruits. In addition, the expression patterns of CmRIN14 and CmRIN15 between the two melon materials presented a opposite trend,indicating that their expression patterns were affected by the expression level of CmACO1.
引文
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[12] Lacey R F,Binder B M.How plants sense ethylene gas-the ethylene receptors[J].Journal of Inorganic Biochemistry,2014,133(2):58-62.doi:10.1016/j.jinorgbio.2014.01.006.
[13] Gao Z,Chen Y F,Randlett M D,Zhao X C,Findell J L,Kieber J J,Schaller G E.Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes[J].Journal of Biological Chemistry,2003,278(36):34725-34732.doi:10.1074/jbc.M305548200.
[14] Zhong S,Lin Z,Grierson D.Tomato ethylene receptor CTR interactions:visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum[J].Journal of Experimental Botany,2008,59(4):965-972.doi:10.1093/jxb/ern021.
[15] 玉庄.转ACC氧化酶反义基因河套蜜瓜耐贮藏品系选育[D].呼和浩特:内蒙古大学,2008.doi:10.7666/d.y1376451.Yu Z.The selection of longer shelf-life strains of Cucumis melo L.cv.Hetao transformed with an antisense ACC oxidase gene[D].Hohhot:Inner Mongolia University,2008.
[16] 田世平.果实成熟和衰老的分子调控机制[J].植物学报,2013,48(5):481-488.doi:10.3724/SP.J.1259.2013.00481.Tian S P.Molecular regulation mechanism of fruit ripening and senescence[J].Chinese Bulletin of Botany,2013,48(5):481-488.
[17] Gapper N E,Mcquinn R P,Giovannoni J J.Molecular and genetic regulation of fruit ripening[J].Plant Molecular Biology,2013,82(6):575-591.doi:10.1007/s11103-013-0050-3.
[18] Seymour G B,Φstergaard L,Chapman N H,Knapp S,Martin C.Fruit development and ripening[J].Annual review of Plant Biology,2013,64(16):219-241.doi:10.1146/annurev-arplant-050312-120057.
[19] 张自强,白晨,张惠忠,李晓东,付增娟,赵尚敏,鄂圆圆,张辉,王良,张必周.转录组测序及其在甜菜功能基因挖掘中的应用[J].北方农业学报,2018,46(5):39-43.doi:10.3969/j.issn.2096-1197.2018.05.06.Zhang Z Q,Bai C,Zhang H Z,Li X D,Fu Z J,Zhao S M,E Y Y,Zhang H,Wang L,Zhang B Z.Transcriptome sequencing and its application in sugar beet functional gene resource discovery[J].Journal of Northern Agriculture,2018,46(5):39-43.
[20] Ciardi J A,Tieman D M,Jones J B.Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv.vesicatoria[J].Molecular plant-microbe interactions,2001,14(4):487-495.doi:10.1094/MPMI.2001.14.4.487.
[21] 姚远,高峰,郝金凤,哈斯阿古拉.甜瓜乙烯信号转导途径关键因子基因CTR1的克隆及表达特性分析[J].生物技术通报,2011(11):83-87.doi:10.13560/j.cnki.biotech.bull.1985.2011.11.004.Yao Y,Gao F,Hao J F,Hasi A.Cloning and expression analysis of a key gene CTR1 in Ethylene Signal transduction in Cucumis melo[J].Biotechnology Bulletin,2011(11):83-87.
[2] Kazan K.Diverse roles of jasmonates and ethylene in abiotic stress tolerance[J].Trends in Plant Science,2015,20(4):219-229.doi:10.1016/j.tplants.2015.02.001.
[3] Mcmurchie E J,Mcglasson W B,Eaks I L.Treatment of fruit with propylene gives information about the biogenesis of ethylene[J].Nature,1972,237(5352):235-236.doi:10.1038/237235a0.
[4] Lelièvre J M,Latchè A,Jones B,Bouzayen M.Ethylene and fruit ripening[J].Physiologia Plantarum,2007,26(2):143-159.doi:10.1002/9781118223086.ch11.
[5] Nakatsuka A,Murachi S,Okunishi H,Shiomi S,Nakano R,Kubo Y,Inaba A.Differential expression and internal feedback regulation of 1-aminocyclopropane-1-Carboxylate synthase,1-aminocyclopropane-1-carboxylate oxidase,and ethylene receptor genes in tomato fruit during development and ripening[J].Plant Physiology,1998,118(4):1295-1305.doi:10.2307/4278560.
[6] Inaba A.Studies on the internal feedback regulation of ethylene biosynthesis and signal transduction during fruit ripening,and the improvement of fruit quality[J].Journal of the Japanese Society for Horticultural Science,2007,76(1):1-12.doi:10.2503/jjshs.76.1.
[7] Barry C S,Lloptous M I,Grierson D.The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[J].Plant Physiology,2000,123(3):979-986.doi:10.2307/4279329.
[8] Naoki Y,Ryohei N,Shunsuke I,Nagata M,Inaba A,Kubo Y.Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated[J].Journal of Experimental Botany,2009,60(12):3433-3442.doi:10.1093/jxb/erp185.
[9] Lin Z,Hong Y,Yin M,Zhang K,Grierson D.A tomato HD-Zip homeobox protein,LeHB-1,plays an important role in floral organogenesis and ripening[J].Plant Journal,2008,55(2):301-310.doi:10.1111/j.1365-313X.2008.03505.x.
[10] Ito Y,Kitagawa M,Ihashi N,Yabe K,Kimbara J,Yasuda J,Ito H,Inakuma T,Hiroi S,Kasumi T.DNA-binding specificity,transcriptional activation potential,and the rin mutation effect for the tomato fruit-ripening regulator RIN[J].Plant Journal,2008,55:212-223.doi:10.1111/j.1365-313X.2008.03491.x.
[11] Vrebalov J,Ruezinsky D,Padmanabhan V,White R,Medrano D,Drake R,Schuch W,Giovannoni J.A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (Rin) locus[J].Science,2002,296:343-346.doi:10.1126/science.1068181.
[12] Lacey R F,Binder B M.How plants sense ethylene gas-the ethylene receptors[J].Journal of Inorganic Biochemistry,2014,133(2):58-62.doi:10.1016/j.jinorgbio.2014.01.006.
[13] Gao Z,Chen Y F,Randlett M D,Zhao X C,Findell J L,Kieber J J,Schaller G E.Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes[J].Journal of Biological Chemistry,2003,278(36):34725-34732.doi:10.1074/jbc.M305548200.
[14] Zhong S,Lin Z,Grierson D.Tomato ethylene receptor CTR interactions:visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum[J].Journal of Experimental Botany,2008,59(4):965-972.doi:10.1093/jxb/ern021.
[15] 玉庄.转ACC氧化酶反义基因河套蜜瓜耐贮藏品系选育[D].呼和浩特:内蒙古大学,2008.doi:10.7666/d.y1376451.Yu Z.The selection of longer shelf-life strains of Cucumis melo L.cv.Hetao transformed with an antisense ACC oxidase gene[D].Hohhot:Inner Mongolia University,2008.
[16] 田世平.果实成熟和衰老的分子调控机制[J].植物学报,2013,48(5):481-488.doi:10.3724/SP.J.1259.2013.00481.Tian S P.Molecular regulation mechanism of fruit ripening and senescence[J].Chinese Bulletin of Botany,2013,48(5):481-488.
[17] Gapper N E,Mcquinn R P,Giovannoni J J.Molecular and genetic regulation of fruit ripening[J].Plant Molecular Biology,2013,82(6):575-591.doi:10.1007/s11103-013-0050-3.
[18] Seymour G B,Φstergaard L,Chapman N H,Knapp S,Martin C.Fruit development and ripening[J].Annual review of Plant Biology,2013,64(16):219-241.doi:10.1146/annurev-arplant-050312-120057.
[19] 张自强,白晨,张惠忠,李晓东,付增娟,赵尚敏,鄂圆圆,张辉,王良,张必周.转录组测序及其在甜菜功能基因挖掘中的应用[J].北方农业学报,2018,46(5):39-43.doi:10.3969/j.issn.2096-1197.2018.05.06.Zhang Z Q,Bai C,Zhang H Z,Li X D,Fu Z J,Zhao S M,E Y Y,Zhang H,Wang L,Zhang B Z.Transcriptome sequencing and its application in sugar beet functional gene resource discovery[J].Journal of Northern Agriculture,2018,46(5):39-43.
[20] Ciardi J A,Tieman D M,Jones J B.Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv.vesicatoria[J].Molecular plant-microbe interactions,2001,14(4):487-495.doi:10.1094/MPMI.2001.14.4.487.
[21] 姚远,高峰,郝金凤,哈斯阿古拉.甜瓜乙烯信号转导途径关键因子基因CTR1的克隆及表达特性分析[J].生物技术通报,2011(11):83-87.doi:10.13560/j.cnki.biotech.bull.1985.2011.11.004.Yao Y,Gao F,Hao J F,Hasi A.Cloning and expression analysis of a key gene CTR1 in Ethylene Signal transduction in Cucumis melo[J].Biotechnology Bulletin,2011(11):83-87.