CNR转录因子在番茄果实成熟过程中的功能

doi:10.13560/j.cnki.biotech.bull.1985.2020-0783

摘要/Abstract

摘要：

SPL转录因子在植物中广泛存在并参与植物生长、发育和成熟过程,CNR是SPL转录因子家族中的一个成员,其作用机制尚不清楚。通过RNA-seq、qRT-PCR和染色质免疫共沉淀技术（ChIP）确定转录因子CNR直接作用的新的靶基因,旨在揭示番茄果实成熟过程中CNR的转录调控网络。通过RNA-seq筛选出野生型AC和突变体Cnr番茄果实中有10 223个差异表达基因,这些基因涉及番茄果实生长和发育的许多方面。qRT-PCR验证的结果和转录组测序数据一致,发现甘露聚糖-1,4-β-甘露糖苷酶基因、亚油酸 9S-脂氧合酶基因、果胶酯酶基因、八氢番茄红素合成酶基因、1-氨基环丙烷-1-羧酸合成酶基因、乙烯响应因子基因、多聚半乳糖醛酸酶基因和果胶裂解酶基因在AC和Cnr番茄果实中的表达量有显著差异。同时,在这些基因的启动子区域发现了转录因子CNR的结合位点GTAC 基序,揭示CNR可能直接调控这些基因的转录,最后通过ChIP分析进一步验证了这个假设。转录因子CNR新靶基因涉及脂代谢、细胞壁、番茄红素合成和乙烯生物合成途径,CNR通过这些途径在番茄果实成熟过程中起作用。

关键词: CNR转录因子, 靶基因, 成熟过程, 番茄果实, ChIP

Abstract:

SPL transcription factors are widely existing in plants and are involved in plant growth,development and maturation. CNR is a member of SPL transcription factor family,and its mechanism of action is still unclear. The novel target genes of CNR were determined by RNA-seq,qRT-PCR and chromatin immunoprecipitation（ChIP）assay,which is aimed to uncover the transcriptional regulatory network of CNR during the ripening process of tomato fruit. About 10,223 differentially expressed genes between wild type AC and mutant cnr were achieved by RNA-seq,and these genes were involved in many aspects of tomato fruit growth and development. The results of qTR-PCR were basically consistent with transcriptome sequenced data. Particularly there were significantly large differential expression levels of gene Mannan endo-1,4-beta-mannosidase,Linoleate 9S-lipoxygenase,Pectinesterase,Phytoene synthase,1-aminocyclopropane-1-carboxylate synthase,Ethylene response factor,Polygalacturonase and Pectate lyase between AC and cnr. In addition,GTAC motif,a CNR-binding site,was found in the promoter regions of these genes,suggesting that CNR possibly directly regulated their transcriptions,and this supposition was further verified by ChIP analysis. Functional annotations of CNR target genes uncovered the specific role of CNR during the ripening process of tomato fruit via the pathways of lipid metabolism,cell wall,lycopene synthesis and ethylene biosynthesis.

Key words: CNR transcription factor, target genes, ripening process, tomato fruit, ChIP

李玲, 杨丽霞, 郭梅. CNR转录因子在番茄果实成熟过程中的功能[J]. 生物技术通报, 2021, 37(2): 51-62.

LI Ling, YANG Li-xia, GUO Mei. Function of Transcription Factor CNR in the Ripening Process of Tomato Fruit[J]. Biotechnology Bulletin, 2021, 37(2): 51-62.

图/表 11

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基因名称/ID	引物名称	引物序列（5'-3'）
Actin	Actin F	CTTCCCTCAGCACCTTCCAG
Actin	Actin R	GCATCTCTGGTCCAGTAGGAA
Solyc01g008710.2	01g008710-F	GTTTCAGGGATTGGATTTTGTG
Solyc01g008710.2	01g008710-R	CATCACTTGTTAATTTCTGCCCTC
Solyc12g005630.1	12g005630-F	GAACCCTCACACAAGGACTAAAG
Solyc12g005630.1	12g005630-R	ACGGCACGACACAACCAAG
Solyc08g014000.2	08g014000-F	CGAGGCGTGGGATAGGATTTA
Solyc08g014000.2	08g014000-R	CTCTCCGAGGATACGGGTAGTC
Solyc01g103920.2	01g103920-F	GCCTATCCACAGTCTGATGTTACC
Solyc01g103920.2	01g103920-R	GCGACAAACAAATCTACAAAACAG
Solyc05g052390.2	05g052390-F	TGGAAGTAAACAACGGGGATA
Solyc05g052390.2	05g052390-R	TCTTTCCTGGTCTAATCGGGC
Solyc07g064180.2	07g064180-F	TCGAGTTGGAGCTGATAAGTCTG
Solyc07g064180.2	07g064180-R	GGAATACAACTGCTGCATTACCG
Solyc02g068590.2	02g068590-F	CTCAGCATTCAGCAGATTACCTAC
Solyc02g068590.2	02g068590-R	TGACTGTGTTGTTGTGCCCTT
novel.9194	novel-9194-F	TCTGAATCACTCAATTCCAATCAGC
novel.9194	novel-9194-R	AGAAGTGAAGGGAAACAAACCT
Solyc05g050010.2	05g050010-F	AACTAGATTCGCCCAGGCTC
Solyc05g050010.2	05g050010-R	AGGCGACACTAAAATCAACATAAA
Solyc06g065820.2	06g065820-F	AATCTCCTTCCTCAACCAATTTCA
Solyc06g065820.2	06g065820-R	ATCTTTGTTGTGCCCTAGCCA
Solyc02g084850.2	02g084850-F	ATTTGCCACGATCCCGCTAA
Solyc02g084850.2	02g084850-R	TGGAGGTTCAGGGAGTAGGG
Solyc02g084870.2	02g084870-F	TGCTTTCGGTTTACTACACTCAG
Solyc02g084870.2	02g084870-R	ATTCCATTCGGAGCCTCCAC
Solyc07g064160.2	07g064160-F	TCTCATCACTCAGCTCTACACT
Solyc07g064160.2	07g064160-R	AGGGCGGAGAAGATTGTGTT
Solyc02g079220.2	02g079220-F	TGGCAAAGCAACTGTGTAGC
Solyc02g079220.2	02g079220-R	TGTATCCAGTGCACAGCTTATG
Solyc01g096190.2	01g096190-F	ATGCCTTGGAAGGCTGCTT
Solyc01g096190.2	01g096190-R	CAGCAACAATAACACTCATTGAGGA
Solyc08g029000.2	08g029000-F	TTCCTAATCACGGCAGCGTT
Solyc08g029000.2	08g029000-R	TCCATCTCCTCTTAAGTTCACTAGC
Solyc02g082090.2	02g082090-F	ACTGCACCAATTGTCACTGC
Solyc02g082090.2	02g082090-R	GGCGTTAACACGGGAACAAG
Solyc09g011630.2	09g011630-F	CCGCCAAGAGATGAAATACTTGC
Solyc09g011630.2	09g011630-R	ACCCACATGCACTCATAAACAA
Solyc01g101190.2	01g101190-F	GCACTTGCTTTCTCCACTGC
Solyc01g101190.2	01g101190-R	TTTGCAAATCGGAGCAGCAC
Solyc09g075330.2	09g075330-F	TTCAAAAACGGACGGTTGCC
Solyc09g075330.2	09g075330-R	CCACTTCCGTCCTGACTCAC
Solyc07g063950.2	07g063950-F	GAAAAGGCGGATGGGAGGAA
Solyc07g063950.2	07g063950-R	CCTCATGGAAGGCCGTTTCT
Solyc12g008980.1	12g008980-F	GAGATTGAAACCGTTGGTCG
Solyc12g008980.1	12g008980-R	ACACAACTGTCACTACCACTACT
Solyc08g080630.2	08g080630-F	AGAAGATGTCAAGTAGCCCATGT
Solyc08g080630.2	08g080630-R	TACATAGTGCCAAAAGCCGC
Solyc07g006900.1	07g006900-F	CGCGCTTTTCATCTGGCATC
Solyc07g006900.1	07g006900-R	GGGATCCCCATAACTAGAGTGT
Solyc01g060020.2	01g060020-F	ATCATACACAAGTCTATCTCAGTGT
Solyc01g060020.2	01g060020-R	TGATGGCAAGTTGTTCCCCA

基因名称/ID	引物名称	引物序列（5'-3'）
Actin	Actin F	CTTCCCTCAGCACCTTCCAG
Actin	Actin R	GCATCTCTGGTCCAGTAGGAA
Solyc01g008710.2	01g008710-F	GTTTCAGGGATTGGATTTTGTG
Solyc01g008710.2	01g008710-R	CATCACTTGTTAATTTCTGCCCTC
Solyc12g005630.1	12g005630-F	GAACCCTCACACAAGGACTAAAG
Solyc12g005630.1	12g005630-R	ACGGCACGACACAACCAAG
Solyc08g014000.2	08g014000-F	CGAGGCGTGGGATAGGATTTA
Solyc08g014000.2	08g014000-R	CTCTCCGAGGATACGGGTAGTC
Solyc01g103920.2	01g103920-F	GCCTATCCACAGTCTGATGTTACC
Solyc01g103920.2	01g103920-R	GCGACAAACAAATCTACAAAACAG
Solyc05g052390.2	05g052390-F	TGGAAGTAAACAACGGGGATA
Solyc05g052390.2	05g052390-R	TCTTTCCTGGTCTAATCGGGC
Solyc07g064180.2	07g064180-F	TCGAGTTGGAGCTGATAAGTCTG
Solyc07g064180.2	07g064180-R	GGAATACAACTGCTGCATTACCG
Solyc02g068590.2	02g068590-F	CTCAGCATTCAGCAGATTACCTAC
Solyc02g068590.2	02g068590-R	TGACTGTGTTGTTGTGCCCTT
novel.9194	novel-9194-F	TCTGAATCACTCAATTCCAATCAGC
novel.9194	novel-9194-R	AGAAGTGAAGGGAAACAAACCT
Solyc05g050010.2	05g050010-F	AACTAGATTCGCCCAGGCTC
Solyc05g050010.2	05g050010-R	AGGCGACACTAAAATCAACATAAA
Solyc06g065820.2	06g065820-F	AATCTCCTTCCTCAACCAATTTCA
Solyc06g065820.2	06g065820-R	ATCTTTGTTGTGCCCTAGCCA
Solyc02g084850.2	02g084850-F	ATTTGCCACGATCCCGCTAA
Solyc02g084850.2	02g084850-R	TGGAGGTTCAGGGAGTAGGG
Solyc02g084870.2	02g084870-F	TGCTTTCGGTTTACTACACTCAG
Solyc02g084870.2	02g084870-R	ATTCCATTCGGAGCCTCCAC
Solyc07g064160.2	07g064160-F	TCTCATCACTCAGCTCTACACT
Solyc07g064160.2	07g064160-R	AGGGCGGAGAAGATTGTGTT
Solyc02g079220.2	02g079220-F	TGGCAAAGCAACTGTGTAGC
Solyc02g079220.2	02g079220-R	TGTATCCAGTGCACAGCTTATG
Solyc01g096190.2	01g096190-F	ATGCCTTGGAAGGCTGCTT
Solyc01g096190.2	01g096190-R	CAGCAACAATAACACTCATTGAGGA
Solyc08g029000.2	08g029000-F	TTCCTAATCACGGCAGCGTT
Solyc08g029000.2	08g029000-R	TCCATCTCCTCTTAAGTTCACTAGC
Solyc02g082090.2	02g082090-F	ACTGCACCAATTGTCACTGC
Solyc02g082090.2	02g082090-R	GGCGTTAACACGGGAACAAG
Solyc09g011630.2	09g011630-F	CCGCCAAGAGATGAAATACTTGC
Solyc09g011630.2	09g011630-R	ACCCACATGCACTCATAAACAA
Solyc01g101190.2	01g101190-F	GCACTTGCTTTCTCCACTGC
Solyc01g101190.2	01g101190-R	TTTGCAAATCGGAGCAGCAC
Solyc09g075330.2	09g075330-F	TTCAAAAACGGACGGTTGCC
Solyc09g075330.2	09g075330-R	CCACTTCCGTCCTGACTCAC
Solyc07g063950.2	07g063950-F	GAAAAGGCGGATGGGAGGAA
Solyc07g063950.2	07g063950-R	CCTCATGGAAGGCCGTTTCT
Solyc12g008980.1	12g008980-F	GAGATTGAAACCGTTGGTCG
Solyc12g008980.1	12g008980-R	ACACAACTGTCACTACCACTACT
Solyc08g080630.2	08g080630-F	AGAAGATGTCAAGTAGCCCATGT
Solyc08g080630.2	08g080630-R	TACATAGTGCCAAAAGCCGC
Solyc07g006900.1	07g006900-F	CGCGCTTTTCATCTGGCATC
Solyc07g006900.1	07g006900-R	GGGATCCCCATAACTAGAGTGT
Solyc01g060020.2	01g060020-F	ATCATACACAAGTCTATCTCAGTGT
Solyc01g060020.2	01g060020-R	TGATGGCAAGTTGTTCCCCA

Sample	Raw Reads	Clean Reads	Clean Bases	Error/%	Q20/%	Q30/%	GC Content/%
AC_1	44167720	44167720	6.63G	0.03	96.21	90.56	41.70
AC_2	46900412	46900412	7.04G	0.03	95.33	90.31	42.14
AC_3	53236492	53236492	7.99G	0.03	95.47	90.52	42.04
Cnr_1	56396376	56396376	8.46G	0.03	96.06	90.32	41.62
Cnr_2	53171314	53171314	7.98G	0.03	95.30	90.29	41.63
Cnr_3	47154954	47154954	7.07G	0.04	94.18	88.92	41.59

Sample	Raw Reads	Clean Reads	Clean Bases	Error/%	Q20/%	Q30/%	GC Content/%
AC_1	44167720	44167720	6.63G	0.03	96.21	90.56	41.70
AC_2	46900412	46900412	7.04G	0.03	95.33	90.31	42.14
AC_3	53236492	53236492	7.99G	0.03	95.47	90.52	42.04
Cnr_1	56396376	56396376	8.46G	0.03	96.06	90.32	41.62
Cnr_2	53171314	53171314	7.98G	0.03	95.30	90.29	41.63
Cnr_3	47154954	47154954	7.07G	0.04	94.18	88.92	41.59

Sample	Total reads	Total mapped	% of reads mapped	Uniquely mapped	% of uniquely mapped	Splice reads
AC_1	44167720	41773659	94.58	41185239	93.25	16180308
AC_2	46900412	44392160	94.65	43756611	93.30	17617378
AC_3	53236492	50451773	94.77	49710624	93.38	19968698
Cnr_1	56396376	52928267	93.85	51634300	91.56	18711319
Cnr_2	53171314	49935129	93.91	48982327	92.12	18216094
Cnr_3	47154954	43817610	92.92	42981026	91.15	16053453