生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 147-160.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0746
吕宇婧1(), 吴丹丹1, 孔春艳1,2, 杨宇1, 龚明1()
收稿日期:
2022-06-21
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
吕宇婧,女,硕士研究生,研究方向:生物化学与分子生物学;E-mail: 基金资助:
LV Yu-jing1(), WU Dan-dan1, KONG Chun-yan1,2, YANG Yu1, GONG Ming1()
Received:
2022-06-21
Published:
2023-02-26
Online:
2023-03-07
摘要:
小桐子(Jatropha curcas L.)是极具开发潜力的多用途能源植物,也是喜温冷敏植物,低温严重影响其生长和开发利用。已知木葡聚糖内转糖苷酶/水解酶(XTH)作为细胞壁的修饰酶参与植物对逆境胁迫的响应与适应。前期研究表明12℃冷锻炼可显著提高小桐子抗冷性,而XTH基因对冷锻炼过程高响应。本文基于小桐子低温锻炼期间的转录组、miRNA组和降解组数据,在全基因组层面上对小桐子XTH基因家族进行了鉴定、生物信息学分析、表达谱分析及互作miRNAs的鉴定和分析。结果表明,小桐子中鉴定到XTH基因家族成员29个,编码269-341个氨基酸,其蛋白主要定位于细胞壁,结构均具有Glyco_hyho_16和XET_C结构域以及保守位点ExDxE。29个家族成员可分为3个组别,定位于9条染色体上,其中存在8对基因重复事件。对小桐子植株不同器官、低温锻炼过程及干旱和盐胁迫下的表达谱及RT-qPCR分析显示,该基因家族在不同情况下各成员存在显著的差异化表达模式,表明该基因家族不同成员的作用不尽相同。对降解组和miRNA组测序数据分析结果表明,68个miRNAs靶向调控JcXTH基因家族中的26个成员;选取低温锻炼下表达显著的miRNAs进行表达谱分析,并与其靶向的JcXTHs的基因表达进行相关性分析,显示二者之间呈现显著的负相关,表明在低温锻炼下miRNAs负调控JcXTHs基因的表达。研究结果有助于解析小桐子JcXTH基因家族的功能,以及靶向该家族基因的miRNAs与JcXTH基因互作如何来参与小桐子对低温胁迫的适应,从而为后续通过分子育种改良小桐子抗冷性提供参考。
吕宇婧, 吴丹丹, 孔春艳, 杨宇, 龚明. 小桐子XTH基因家族和与之互作的miRNAs的全基因组鉴定及其在低温适应中的作用[J]. 生物技术通报, 2023, 39(2): 147-160.
LV Yu-jing, WU Dan-dan, KONG Chun-yan, YANG Yu, GONG Ming. Genome-wide Identification of XTH Gene Family and Their Interacting miRNAs and Possible Roles in Low Temperature Adaptation in Jatropha curcas L.[J]. Biotechnology Bulletin, 2023, 39(2): 147-160.
引物名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
JcXTH2-F | GAGAGCAACAATTCCACCTTTG |
JcXTH2-R | GGGTAAGGAACACCGTTTGA |
JcXTH3-F | CTCCCTTCACTGCTTCTTACAG |
JcXTH3-R | CACCCATTTGAGCCTCTCTT |
JcXTH18-F | CTACCAGGGAATACCACACTTAC |
JcXTH18-R | CGTTCCACAGGCTTGAGTATAA |
JcXTH19-F | GTGCACAAGAACAATGGAGAAC |
JcXTH19-R | TGACAACCATCGACGCTAAA |
JcXTH23-F | GCAACAAGAGGTGGACTAGAA |
JcXTH23-R | CATGGCACTTAGGGCTTGATA |
JcXTH27-F | TACTGTTGGGCGCGATAATG |
JcXTH27-R | CCAGACCCAGAAACCCTATCTA |
GAPDH-F | TGAAGGACTGGAGAGGTGGAAGAGC |
GAPDH-R | ATCAACAGTTGGAACACGGAAAGCC |
表1 RT-qPCR分析相关引物
Table 1 Related primers for RT-qPCR analysis
引物名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
JcXTH2-F | GAGAGCAACAATTCCACCTTTG |
JcXTH2-R | GGGTAAGGAACACCGTTTGA |
JcXTH3-F | CTCCCTTCACTGCTTCTTACAG |
JcXTH3-R | CACCCATTTGAGCCTCTCTT |
JcXTH18-F | CTACCAGGGAATACCACACTTAC |
JcXTH18-R | CGTTCCACAGGCTTGAGTATAA |
JcXTH19-F | GTGCACAAGAACAATGGAGAAC |
JcXTH19-R | TGACAACCATCGACGCTAAA |
JcXTH23-F | GCAACAAGAGGTGGACTAGAA |
JcXTH23-R | CATGGCACTTAGGGCTTGATA |
JcXTH27-F | TACTGTTGGGCGCGATAATG |
JcXTH27-R | CCAGACCCAGAAACCCTATCTA |
GAPDH-F | TGAAGGACTGGAGAGGTGGAAGAGC |
GAPDH-R | ATCAACAGTTGGAACACGGAAAGCC |
基因名称Gene name | 基因ID Gene ID | 氨基酸数目 Number of amino acids/aa | 等电点Isoelectric point | 分子量 Molecular weight/kD | 亲水性平均值 GRAVY | 亚细胞定位 Subcellular localization |
---|---|---|---|---|---|---|
JcXTH1 | XM_012218696.1 | 285 | 6.42 | 32.36735 | -0.394 | 细胞壁, 细胞质 |
JcXTH2 | XM_012218694.1 | 269 | 9.04 | 30.94703 | -0.438 | 细胞壁, 细胞质 |
JcXTH3 | XM_012218698.1 | 286 | 6.14 | 32.08499 | -0.319 | 细胞壁, 细胞质 |
JcXTH4 | XM_012218697.1 | 286 | 5.47 | 32.20504 | -0.353 | 细胞壁, 细胞质 |
JcXTH5 | XM_012218699.1 | 283 | 5.21 | 31.86363 | -0.405 | 细胞壁, 细胞质 |
JcXTH6 | XM_012218701.1 | 283 | 5.1 | 31.8406 | -0.353 | 细胞壁, 细胞质 |
JcXTH7 | XM_012218700.1 | 283 | 5.18 | 31.72839 | -0.367 | 细胞壁, 细胞质 |
JcXTH8 | XM_012210833.1 | 283 | 5.79 | 31.90475 | -0.296 | 细胞壁, 细胞质 |
JcXTH9 | XM_012218702.1 | 299 | 5.43 | 34.08454 | -0.219 | 细胞壁, 细胞质 |
JcXTH10 | XM_012210835.1 | 279 | 7.61 | 31.34916 | -0.36 | 细胞壁, 细胞质 |
JcXTH11 | XM_012222502.1 | 288 | 8.78 | 32.76008 | -0.301 | 细胞壁, 细胞质 |
JcXTH12 | XM_012227056.1 | 294 | 9.05 | 33.9776 | -0.386 | 细胞壁 |
JcXTH13 | XM_012209320.1 | 293 | 6.64 | 32.91467 | -0.514 | 细胞壁 |
JcXTH14 | XM_012216981.1 | 323 | 6.76 | 36.95264 | -0.393 | 细胞壁 |
JcXTH15 | XM_012225387.1 | 341 | 8.73 | 39.63884 | -0.51 | 细胞壁 |
JcXTH16 | XM_012229959.1 | 311 | 6.41 | 34.99533 | -0.326 | 细胞壁 |
JcXTH17 | XM_012233283.1 | 293 | 8.45 | 34.01056 | -0.452 | 细胞壁, 细胞质 |
JcXTH18 | XM_012212020.1 | 293 | 6.99 | 34.10647 | -0.439 | 细胞壁, 细胞质 |
JcXTH19 | XM_012236012.1 | 299 | 5 | 34.8449 | -0.6 | 细胞壁 |
JcXTH20 | XM_012218715.1 | 269 | 6.31 | 30.87093 | -0.361 | 细胞壁 |
JcXTH21 | XM_012230444.1 | 284 | 6.44 | 32.68983 | -0.361 | 细胞壁 |
JcXTH22 | XM_012215879.1 | 293 | 4.88 | 32.97573 | -0.353 | 细胞壁 |
JcXTH23 | XM_012224087.1 | 292 | 6.7 | 33.62413 | -0.298 | 细胞壁 |
JcXTH24 | XM_012223597.1 | 288 | 5.37 | 32.77481 | -0.297 | 细胞壁 |
JcXTH25 | XM_012217377.1 | 294 | 8.89 | 34.70349 | -0.382 | 细胞壁 |
JcXTH26 | XM_012221356.1 | 291 | 8.51 | 33.28685 | -0.225 | 细胞壁 |
JcXTH27 | XM_012223052.1 | 277 | 8.82 | 31.58769 | -0.288 | 细胞壁, 细胞质 |
JcXTH28 | XM_012219356.1 | 291 | 8.57 | 33.20157 | -0.282 | 细胞壁, 细胞质 |
JcXTH29 | XM_012237054.1 | 288 | 9.03 | 32.9001 | -0.427 | 细胞壁 |
表2 已鉴定的小桐子XTH蛋白的理化性质和亚细胞定位
Table 2 Physicochemical properties and subcellular location of identified XTH proteins in J. curcas
基因名称Gene name | 基因ID Gene ID | 氨基酸数目 Number of amino acids/aa | 等电点Isoelectric point | 分子量 Molecular weight/kD | 亲水性平均值 GRAVY | 亚细胞定位 Subcellular localization |
---|---|---|---|---|---|---|
JcXTH1 | XM_012218696.1 | 285 | 6.42 | 32.36735 | -0.394 | 细胞壁, 细胞质 |
JcXTH2 | XM_012218694.1 | 269 | 9.04 | 30.94703 | -0.438 | 细胞壁, 细胞质 |
JcXTH3 | XM_012218698.1 | 286 | 6.14 | 32.08499 | -0.319 | 细胞壁, 细胞质 |
JcXTH4 | XM_012218697.1 | 286 | 5.47 | 32.20504 | -0.353 | 细胞壁, 细胞质 |
JcXTH5 | XM_012218699.1 | 283 | 5.21 | 31.86363 | -0.405 | 细胞壁, 细胞质 |
JcXTH6 | XM_012218701.1 | 283 | 5.1 | 31.8406 | -0.353 | 细胞壁, 细胞质 |
JcXTH7 | XM_012218700.1 | 283 | 5.18 | 31.72839 | -0.367 | 细胞壁, 细胞质 |
JcXTH8 | XM_012210833.1 | 283 | 5.79 | 31.90475 | -0.296 | 细胞壁, 细胞质 |
JcXTH9 | XM_012218702.1 | 299 | 5.43 | 34.08454 | -0.219 | 细胞壁, 细胞质 |
JcXTH10 | XM_012210835.1 | 279 | 7.61 | 31.34916 | -0.36 | 细胞壁, 细胞质 |
JcXTH11 | XM_012222502.1 | 288 | 8.78 | 32.76008 | -0.301 | 细胞壁, 细胞质 |
JcXTH12 | XM_012227056.1 | 294 | 9.05 | 33.9776 | -0.386 | 细胞壁 |
JcXTH13 | XM_012209320.1 | 293 | 6.64 | 32.91467 | -0.514 | 细胞壁 |
JcXTH14 | XM_012216981.1 | 323 | 6.76 | 36.95264 | -0.393 | 细胞壁 |
JcXTH15 | XM_012225387.1 | 341 | 8.73 | 39.63884 | -0.51 | 细胞壁 |
JcXTH16 | XM_012229959.1 | 311 | 6.41 | 34.99533 | -0.326 | 细胞壁 |
JcXTH17 | XM_012233283.1 | 293 | 8.45 | 34.01056 | -0.452 | 细胞壁, 细胞质 |
JcXTH18 | XM_012212020.1 | 293 | 6.99 | 34.10647 | -0.439 | 细胞壁, 细胞质 |
JcXTH19 | XM_012236012.1 | 299 | 5 | 34.8449 | -0.6 | 细胞壁 |
JcXTH20 | XM_012218715.1 | 269 | 6.31 | 30.87093 | -0.361 | 细胞壁 |
JcXTH21 | XM_012230444.1 | 284 | 6.44 | 32.68983 | -0.361 | 细胞壁 |
JcXTH22 | XM_012215879.1 | 293 | 4.88 | 32.97573 | -0.353 | 细胞壁 |
JcXTH23 | XM_012224087.1 | 292 | 6.7 | 33.62413 | -0.298 | 细胞壁 |
JcXTH24 | XM_012223597.1 | 288 | 5.37 | 32.77481 | -0.297 | 细胞壁 |
JcXTH25 | XM_012217377.1 | 294 | 8.89 | 34.70349 | -0.382 | 细胞壁 |
JcXTH26 | XM_012221356.1 | 291 | 8.51 | 33.28685 | -0.225 | 细胞壁 |
JcXTH27 | XM_012223052.1 | 277 | 8.82 | 31.58769 | -0.288 | 细胞壁, 细胞质 |
JcXTH28 | XM_012219356.1 | 291 | 8.57 | 33.20157 | -0.282 | 细胞壁, 细胞质 |
JcXTH29 | XM_012237054.1 | 288 | 9.03 | 32.9001 | -0.427 | 细胞壁 |
图2 小桐子JcXTH基因家族成员染色体定位及重复事件分析 重复基因对用线条进行连接
Fig. 2 Chromosome location and repeat event analysis of JcXTH gene family members of J. curcas Repeated pairs of genes are linked by lines
图7 JcXTH基因在小桐子不同器官中(A)以及在盐和干旱胁迫下(B)的表达谱 S:盐胁迫;D:干旱胁迫;R:根;L:叶
Fig. 7 Expression profiles of JcXTH genes in different organs(A)and under salt and drought stress(B) S: Salt stress; D: drought stress; R: root; L: leaf
图8 小桐子JcXTH基因家族中响应冷锻炼基因在12℃冷锻炼期间的RT-qPCR分析 不同小写字母表示差异显著(P<0.05)
Fig. 8 RT-qPCR analysis of the genes responding to chill-hardening in JcXTH gene family of J. curcas during chill-hardening at 12℃ Different lowercase letters on the histogram indicate significant differences(P<0.05)
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