水稻OsJMJ718基因可选择性多聚腺苷酸化序列的 克隆及生殖发育期表达模式

doi:10.11983/CBB17114

植物学报 ›› 2018, Vol. 53 ›› Issue (5): 594-602.DOI: 10.11983/CBB17114 cstr: 32102.14.CBB17114

水稻OsJMJ718基因可选择性多聚腺苷酸化序列的克隆及生殖发育期表达模式

鲁丹, 王丽, 宋凡, 陶菊红, 张大兵, 袁政^*()

上海交通大学生命科学技术学院, 上海 200240

收稿日期:2017-06-07 接受日期:2017-10-07 出版日期:2018-09-01 发布日期:2018-11-29
通讯作者: 袁政
作者简介: 作者简介：路安民(图中左), 植物系统分类学家。20世纪60-70年代编著《中国植物志》等, 后从事植物系统发育和进化研究。“七五”以来主持了4项中科院、国家自然科学基金委重大和重点项目。1991年获国务院颁发的有突出贡献科学家荣誉证书。1987年8月-1990年12月担任中科院植物所所长。
基金资助:
国家七大农作物育种专项(No.2016YFD0101107)、上海市科委基础研究重大重点项目(No.14JC1403901)和国家自然科学基金(No.31470397, No.31671260)

Cloning and Expression Pattern Analysis of Rice OsJMJ718 Alternative Polyadenylation Sequences During Reproductive Developmental Stage

Lu Dan, Wang Li, Song Fan, Tao Juhong, Zhang Dabing, Yuan Zheng^*()

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2017-06-07 Accepted:2017-10-07 Online:2018-09-01 Published:2018-11-29
Contact: Yuan Zheng
About author:† These authors contributed equally to this paper

摘要/Abstract

摘要： 可选择性多聚腺苷酸化是真核生物重要的基因调控机制之一, 通过形成不同长度的3'端非翻译区影响信使RNA的稳定性、定位和翻译效率, 从而增加转录本的复杂度。已有研究表明, 拟南芥(Arabidopsis thaliana)中参与DNA去甲基化调控IBM1基因的可选择性多聚腺苷酸化加工受染色质调节因子EDM2调控, 从而影响拟南芥基因组数千基因的CHG甲基化水平, 但该类调控机制是否在其它物种中同样存在仍然未知。以水稻(Oryza sativa)基因组中IBM1同源基因OsJMJ718为研究对象, 利用生物信息学分析和3'RACE实验, 发现IBM1同源基因也存在可选择性多聚腺苷酸化修饰, OsJMJ718基因可能存在9个可选择性多聚腺苷酸化序列。序列比对分析表明, NCBI网站现存日本晴OsJMJ718基因组3'末端序列与9522和明辉63等其它生态型基因组序列组成可能不同。荧光实时定量PCR分析表明, OsJMJ718的9个转录本在水稻生殖发育阶段呈现不同的动态表达模式, 其中TVX5转录本表达量最高。研究获得的OsJMJ718基因可选择性多聚腺苷酸化序列信息及相关的表达模式分析为进一步揭示水稻OsJMJ718基因的可选择性多聚腺苷酸化分子机制和生物学功能奠定了基础。

关键词: 水稻, OsJMJ718, 可选择性多聚腺苷酸化, 3'RACE, 表达模式

Abstract: Alternative polyadenylation (APA) is one of the important regulatory pathways of eukaryotic gene expression. By forming different lengths of 3' untranslated regions, APA affects the stability, localization and translation efficiency of mRNA and increases the complexity of transcripts. The expression of the Arabidopsis gene increased polyadenylation of BONSAI methylation 1 (IBM1) is regulated by chromatin regulatory factor enhanced downy mildew 2 (EDM2), which can further affect the CHG methylation level of the Arabidopsis thaliana genome. However, whether such regulatory mechanisms exist in other species is unknown. To answer this question, we selected OsJMJ718, an IBM1 homologous gene of rice, for research. By using bioinformatics analysis and 3'RACE experiments, we found that homologous genes of IBM1 also had APA modification. Among them, the OsJMJ718 gene may have 9 alternative polyadenylation sequences. Further sequence alignment analysis revealed that the 3' terminal sequence of OsJMJ718 in the existing Japonica genome in the NCBI database may be different from that of the other ecotype genome sequences, such as 9522 and Minghui 63. Quantitative real-time PCR showed that the 9 transcripts of OsJMJ718 present diverse dynamic expression patterns in different stages of rice reproductive development. The expression of OsJMJ718-TVX5 was higher than that of other transcripts. In summary, this work provides information for APA sequences of OsJMJ718, and the expression pattern analysis of these transcripts would also help further study of the mechanism of APA and biological function of OsJMJ718.

Key words: rice, OsJMJ718, alternative polyadenylation, 3'RACE, expression pattern

鲁丹, 王丽, 宋凡, 陶菊红, 张大兵, 袁政. 水稻OsJMJ718基因可选择性多聚腺苷酸化序列的克隆及生殖发育期表达模式. 植物学报, 2018, 53(5): 594-602.

Lu Dan, Wang Li, Song Fan, Tao Juhong, Zhang Dabing, Yuan Zheng. Cloning and Expression Pattern Analysis of Rice OsJMJ718 Alternative Polyadenylation Sequences During Reproductive Developmental Stage. Chinese Bulletin of Botany, 2018, 53(5): 594-602.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB17114

https://www.chinbullbotany.com/CN/Y2018/V53/I5/594

图/表 4

表1 本研究所用引物

Table 1 Primers used in this research

Primer name	Primer sequence (5′-3′)	Type
qRT-F-TVX1	GAGCTTGGATAGCCCGCCTC	qRT PCR
qRT-R-TVX1	TCTTTTCTTCCCGGGAGTGC	qRT PCR
qRT-F-TVX2	CAATAATTGAACTCTAGGTC	qRT PCR
qRT-R-TVX2	TAAGGAAATACAATCAGATC	qRT PCR
qRT-F-TVX3	ACGGACGCTGGATCGGCGAG	qRT PCR
qRT-R-TVX3	TAACAAGAGCAGTAGAGCAC	qRT PCR
qRT-F-TVX4	ACGGACGCTGGATCGGCGAG	qRT PCR
qRT-R-TVX4	AAGGACGGGGATGCGGCGT	qRT PCR
qRT-F-TVX5	AACGACAACTTTAGGGTTCG	qRT PCR
qRT-R-TVX5	TCGTTACAAGAAAGATGAAC	qRT PCR
qRT-F-TVX6	ATCGAATTGCCACGTAAGCG	qRT PCR
qRT-R-TVX6	TCATCCTCACTCTCTTCTTC	qRT PCR
qRT-F-TVX7	GAACCACAGGGCCAAAGAAG	qRT PCR
qRT-R-TVX7	TAATCCAATTAAAAGTGTTG	qRT PCR
qRT-F-TVX9	AACTCTTCACCACGCGTATG	qRT PCR
qRT-R-TVX9	TAACCGGCGATGGCTGCATC	qRT PCR
qRT-F-TVX11	GAATAAGATGATAATCTATG	qRT PCR
qRT-R-TVX11	ATATCTCTAACTCTACATGC	qRT PCR
3'RACE-F-OsJMJ718	GATTACGCCAAGCTTAGTGAGACCAACAAGGGAGGTGCT	3'RACE

图1 拟南芥和水稻中JHDM2家族基因mRNA可选择性多聚腺苷酸化加工序列数目Os09g22540 (JMJ718)为研究对象OsJMJ718; At3g07610 (JMJ25)是拟南芥IBM1, OsJMJ718的同源基因。

Figure 1 The number of alternative polyadenylation sequences of JHDM2 family genes in Arabidopsis thaliana and riceOs09g22540 (JMJ718) is OsJMJ718, the target sequence of this study; At3g07610 (JMJ25) is IBM1, which is the homo- logous gene of OsJMJ718 in Arabidopsis thaliana.

图2 OsJMJ718-TVX转录本序列9个转录本分别命名为TVX1-TVX9; Exon1-Exon10表示各转录本共有外显子序列, Exon11-Exon18为不同颜色表示基因组上的8个外显子通过可选择性多聚腺苷酸化被特异加工至TVX1-TVX7转录本。其中, TVX8和TVX9的3'端序列仅存在于明辉63 (MH63)基因组中。* 表示终止密码子

Figure 2 Sequence analysis of OsJMJ718-TVX transcriptsNine transcripts were named as TVX1-TVX9, respectively; Exon1-Exon10 represent common exon sequences for each transcript; Exon11-Exon18 were marked in different color, the 8 exons were processed in TVX1-TVX7 by alternative polyadenylation. However, 3’ terminal sequences of TVX8 and TVX9 are only exist in MH63 genomic sequences. * indicates stop codon

图3 OsJMJ718-TVX转录本表达模式

Figure 3 Expression pattern analysis of OsJMJ718-TVX transcripts

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水稻OsJMJ718基因可选择性多聚腺苷酸化序列的克隆及生殖发育期表达模式

Cloning and Expression Pattern Analysis of Rice OsJMJ718 Alternative Polyadenylation Sequences During Reproductive Developmental Stage

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水稻OsJMJ718基因可选择性多聚腺苷酸化序列的 克隆及生殖发育期表达模式

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水稻OsJMJ718基因可选择性多聚腺苷酸化序列的克隆及生殖发育期表达模式