小麦TaNAC基因基于可变剪切和microRNA的转录后调控分析

doi:10.3864/j.issn.0578-1752.2021.22.001

摘要/Abstract

摘要：

【目的】以条锈菌和白粉菌胁迫的普通小麦（Triticum aestivum L.）为研究对象,分析由可变剪切（alternative splicing,AS）形成的TaNAC结构变异转录本,同时分析TaNAC基因的microRNA调控位点,为进一步解析TaNAC基因通过转录后调控参与小麦响应真菌胁迫奠定基础。【方法】普通小麦兼抗种质N9134在被白粉菌和条锈菌分别侵染后,各8个时间点取样并混合,然后从混合样本中克隆得到大量TaNAC转录本。参考中国春小麦基因组注释信息（IWGSC RefSeq v1.1）进行比对,选择由可变剪切形成的TaNAC序列结构变异转录本,分析它们的序列结构特征。利用生物信息学软件和在线工具,对这些TaNAC结构变异转录本编码产物的功能结构域、高级结构、理化性质、亚细胞定位等特征和变异情况进行比对分析。同时,利用洋葱表皮细胞瞬时表达系统验证其中1对TaNAC结构变异转录本的亚细胞定位预测结果,并选取5组TaNAC基因的可变剪切序列结构变异转录本进行酵母转录自激活试验,研究序列结构变异对TaNAC基因转录调控活性的影响。此外,利用miRBase数据库收录的小麦中已报道的miRNAs和TaNAC基因,进行靶基因预测分析,建立小麦TaNAC家族成员与miRNAs的靶向关系。【结果】以条锈菌和白粉菌侵染的普通小麦兼抗种质N9134为材料,克隆得到的TaNAC转录本中的35条序列结构变异转录本由13个TaNAC基因可变剪切形成。通过分析发现,同一TaNAC基因由可变剪切形成的不同结构变异转录本的核酸序列结构存在差异,而且其对应编码产物的功能结构域、高级结构、理化性质和亚细胞定位等方面均会存在差异,同时也会表现出不同的转录调控活性;不同TaNAC基因的可变剪切方式存在差异,而且它们的结构变异转录本及其编码产物在结构特征、理化性质和转录调控活性等方面也均呈现出多样性的特征。通过分析TaNAC基因与其在编码序列区域的靶标tae-miRNAs,发现具有可变剪切转录本的TaNAC基因与tae-miRNA的结合位点均在其非可变剪切区域。【结论】TaNAC基因可以通过可变剪切这种转录后调控方式参与小麦对真菌胁迫的响应;同时发现,调控TaNAC基因的tae-miRNA可以独立于可变剪切这种转录后调控方式行使功能。

关键词: 小麦, TaNAC转录因子, microRNA, 可变剪切, 转录后调控, 胁迫响应

Abstract:

【Objective】 In the present study, the common wheat (Triticum aestivum L.) was exposed to the stress of stripe rust and powdery mildew. Then the cloned TaNAC structural variation transcripts formed by alternative splicing were analyzed. And putative information of TaNAC genes regulated by miRNAs was annotated. It would shed light on the study of TaNAC genes in response to fungal stress of wheat at the post-transcriptional level. 【Method】 After common wheat resistance germplasm N9134 being infected stripe rust and powdery mildew respectively, the leaves were sampled at eight time points. Then a large number of TaNAC transcripts were cloned from the mixed sample pool. Referring to the wheat genome annotation of Chinese Spring (IWGSC RefSeqv1.1), the sequence structure characteristics of TaNAC structural variation transcripts formed by alternative cutting were revealed. Using bioinformatics software and online tools, the coding products derived from these TaNAC transcripts were compared and analyzed, including the functional domain, advanced structure, physical and chemical properties, subcellular localization and other characteristics. And then, one pair of TaNAC structural variation transcripts were selected to further verify the predicted subcellular localizations by onion epidermis transient expression system. Meanwhile, five groups of TaNAC transcripts were conducted transcriptional self-activation experiments in yeast. It was aimed to analyze the effects of structural variation, which caused by alternative splicing on transcriptional regulation activity. Additionally, using the miRBase database, the targeting relationship between TaNAC genes and tae-miRNAs was forecasted and established in wheat. 【Result】 In this study, 35 TaNAC structural variation transcripts were formed by alternative splicing form 13 TaNAC genes, and they all were cloned from common wheat N9134 after infecting by stripe rust and powdery mildew. After analyzing, it was found that there were differences in the nucleic acid sequence structure of different structural variation transcripts from the same TaNAC gene, as well as in the functional domain, advanced structure, physicochemical properties and subcellular localization of their corresponding coding products. And they might be with different transcriptional regulatory activities. Moreover, different TaNAC genes could be with different patterns of alternative splicing, and the coded products of the structural variation transcripts from different TaNAC genes showed diversity in characteristics of structure, physical and chemical properties, transcriptional regulatory activity and so on. By analysis of TaNAC genes and their target tae-miRNAs, which is in the coding region, the result showed that the binding sites of tae-miRNAs were all in the non-alternative splicing region. 【Conclusion】 In conclusion, TaNAC genes might be involved in the response of wheat to fungal stress through the post-transcriptional regulation of alternative splicing. And the tae-miRNAs targeted to TaNAC genes could function post-transcriptional regulation independently of alternative splicing.

Key words: wheat, TaNAC transcription factor, microRNA, alternative splicing, post-transcriptional regulation, stress responses

吕士凯, 马小龙, 张敏, 邓平川, 陈春环, 张宏, 刘新伦, 吉万全. 小麦TaNAC基因基于可变剪切和microRNA的转录后调控分析[J]. 中国农业科学, 2021, 54(22): 4709-4727.

LÜ ShiKai, MA XiaoLong, ZHANG Min, DENG PingChuan, CHEN ChunHuan, ZHANG Hong, LIU XinLun, JI WanQuan. Post-transcriptional Regulation of TaNAC Genes by Alternative Splicing and MicroRNA in Common Wheat (Triticum aestivum L.)[J]. Scientia Agricultura Sinica, 2021, 54(22): 4709-4727.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.22.001

https://www.chinaagrisci.com/CN/Y2021/V54/I22/4709

图/表 14

表1

图1

图2

表2

克隆得到的13个基因可变剪切形成的35条TaNAC结构变异转录本"

分组 Groups	TaNAC转录本名称 Names of TaNAC transcripts	序列长度 Sequence length (bp)	GenBank登录号 Accessions	对应中国春参考基因组的基因 The corresponding genes in IWGSC RefSeq v1.1	是否外显子跳跃 Exon skipping or not	是否内含子保留 Intron retention or not	转录本编码方式 Coding mode of transcripts
1	TaNAC_NTL5_7B.2	1932	MN747301	TraesCS7B02G196900	是 Yes	否 No	正常 N
1	TaNAC_NTL5_7B.3	879	MN747304	TraesCS7B02G196900	是 Yes	否 No	正常 N
2	TaNAC008_3A.1	1446	MN747252	TraesCS3A02G176500	是 Yes	否 No	正常 N
2	TaNAC008_3A.2	597	MN747253	TraesCS3A02G176500	是 Yes	否 No	正常 N
3	TaNAC013_3A.1	1017	MN747260	TraesCS3A02G245900	是 Yes	否 No	正常 N
	TaNAC013_3A.2	1017	MN747262	TraesCS3A02G245900			正常 N
	TaNAC013_3A.3	974	MN786410	TraesCS3A02G245900			提前终止 P
4	TaNAC017_5A.2	2193	MN747191	TraesCS5A02G271500	是 Yes	否 No	正常 N
	TaNAC017_5A.3	1701	MN747193	TraesCS5A02G271500			正常 N
	TaNAC017_5A.4	1181	MN786415	TraesCS5A02G271500			分段 S
	TaNAC017_5A.5	1237	MN786416	TraesCS5A02G271500			分段 S
	TaNAC017_5A.6	1308	MN747194	TraesCS5A02G271500			正常 N
5	TaNAC011_7D.1	1467	MN747256	TraesCS7D02G365200	否 No	是 Yes	正常 N
5	TaNAC011_7D.2	865	MN786437	TraesCS7D02G365200	否 No	是 Yes	分段 S
6	TaNAC017_5D.2	2160	MN747189	TraesCS5D02G279100	否 No	是 Yes	正常 N
6	TaNAC017_5D.4	2052	IWGSC	TraesCS5D02G279100	否 No	是 Yes	正常 N
7	TaNAC024_3D.1	1320	MN747206	TraesCS3D02G109400	否 No	是 Yes	正常 N
	TaNAC024_3D.2	1317	MN747210	TraesCS3D02G109400			正常 N
	TaNAC024_3D.3	1457	MN786432	TraesCS3D02G109400			分段 S
8	TaNAC039_6B.3	1056	MN747281	TraesCS6B02G286200	否 No	是 Yes	正常 N
8	TaNAC039_6B.5	1171	MN786440	TraesCS6B02G286200	否 No	是 Yes	分段 S
9	TaNAC073_3B.3	966	MN747291	TraesCS3B02G410500	否 No	是 Yes	正常 N
	TaNAC073_3B.4	1134	MN786442	TraesCS3B02G410500			分段 S
	TaNAC073_3B.6	979	MN786412	TraesCS3B02G410500			提前终止 P
10	TaNAC092_2A.3	1068	MN747237	TraesCS2A02G338300	否 No	是 Yes	正常 N
10	TaNAC092_2A.5	1325	MN786430	TraesCS2A02G338300	否 No	是 Yes	分段 S
11	TaNAC092_2D.6	1062	MN747248	TraesCS2D02G324700	否 No	是 Yes	正常 N
	TaNAC092_2D.7	1403	MN786434	TraesCS2D02G324700			分段 S
	TaNAC092_2D.8	1405	MN786435	TraesCS2D02G324700			分段 S
12	TaNAC024_3A.1	1320	MN747211	TraesCS3A02G107400	是 Yes	是 Yes	正常 N
	TaNAC024_3A.3	1317	MN747208	TraesCS3A02G107400			正常 N
	TaNAC024_3A.6	1612	MN786431	TraesCS3A02G107400			分段 S
	TaNAC024_3A.7	1401	MN786419	TraesCS3A02G107400			分段 S
13	TaNAC092_2B.1	1065	MN747239	TraesCS2B02G343600	是 Yes	是 Yes	正常 N
	TaNAC092_2B.3	708	MN747234	TraesCS2B02G343600			正常 N
	TaNAC092_2B.7	1167	MN786429	TraesCS2B02G343600			分段 S

表2

图3

表3

克隆得到的13组TaNAC结构变异转录本中正常编码产物的结构特征和理化性质"

分组 Groups	克隆的转录本名称 Name of cloned transcripts	肽链长度 Peptide length (aa)	NAM结构域起止位置 NAM start_end (aa)	跨膜结构域位置 Topology of TMHs	脂肪系数 Aliphatic index	亲水性总平均值 GRAVY	不稳定系数 Instability index	理论等电点 pI	预测的亚细胞定位 Predicted subcellular localization
1	TaNAC_NTL5_7B.2	643	23—149	o620—642i	71.12	-0.424	36.32	4.63	细胞核 Nu
1	TaNAC_NTL5_7B.3	292	无 None	o269—291i	70.45	-0.114	40.41	4.61	胞外/细胞核 Ex / Nu
2	TaNAC008_3A.1	481	10—136	o448—470i	72.45	-0.554	35.92	5.67	细胞核/细胞质 Nu / Cy
2	TaNAC008_3A.2	198	10—136	无 None	65.56	-0.809	37.89	9.08	细胞质 Cy
3	TaNAC013_3A.1	338	73—212	无 None	63.08	-0.656	35.37	8.83	细胞核/线粒体 Nu / Mi
	TaNAC013_3A.2	338	73—212	无 None	63.08	-0.655	35.48	8.83	细胞核/线粒体/细胞质 Nu / Mi / Cy
	TaNAC013_3A.3	266	73—212	无 None	67.18	-0.676	36.58	9.15	细胞核/线粒体 Nu / Mi
4	TaNAC017_5A.2	730	13—136	o695—717i	69.74	-0.511	49.35	4.55	细胞核 Nu
	TaNAC017_5A.3	566	无 None	o531—553i	72.86	-0.403	51.11	4.18	细胞核/胞外 Nu / Ex
	TaNAC017_5A.6	435	13—136	o400—422i	76.00	-0.476	50.17	5.92	细胞核 Nu
	TaNAC017_5A.4	分段 S	— — — — — — — — — — — —
	TaNAC017_5A.5	分段 S	— — — — — — — — — — — —
5	TaNAC011_7D.1	488	57—226	无 None	53.79	-0.843	55.96	7.53	细胞核 Nu
5	TaNAC011_7D.2	分段 S	— — — — — — — — — — — —
6	TaNAC017_5D.2	719	13—136	o683—705i	69.61	-0.490	49.99	4.57	细胞核 Nu
6	TaNAC017_5D.4*	683	13—136	o647—669i	70.26	-0.528	50.36	4.62	细胞核 Nu
7	TaNAC024_3D.1	439	45—186	无 None	59.13	-0.813	48.33	6.88	细胞核 Nu
	TaNAC024_3D.2	438	44—185	无 None	59.27	-0.806	48.24	6.76	细胞核 Nu
	TaNAC024_3D.3	分段 S	— — — — — — — — — — — —
8	TaNAC039_6B.3	351	8—135	无 None	58.97	-0.755	38.99	6.57	细胞核 Nu
8	TaNAC039_6B.5	分段 S	— — — — — — — — — — — —
9	TaNAC073_3B.3	321	46—188	无 None	54.7	-0.902	37.18	8.6	细胞核 Nu
	TaNAC073_3B.6	278	46—188	无 None	59.64	-0.828	33.43	8.83	细胞核 Nu
	TaNAC073_3B.4	分段 S	— — — — — — — — — — — —
10	TaNAC092_2A.3	355	14—138	无 None	64.14	-0.412	30.91	7.21	细胞核/叶绿体 Nu / Ch
10	TaNAC092_2A.5	分段 S	— — — — — — — — — — — —
11	TaNAC092_2D.6	353	14—138	无 None	65.33	-0.397	33.56	6.86	细胞核/叶绿体 Nu / Ch
	TaNAC092_2D.7	分段 S	— — — — — — — — — — — —
	TaNAC092_2D.8	分段 S	— — — — — — — — — — — —
12	TaNAC024_3A.1	439	45—186	无 None	58.45	-0.828	46.99	6.76	细胞核 Nu
	TaNAC024_3A.3	438	44—185	无 None	58.58	-0.821	46.9	6.67	细胞核 Nu
	TaNAC024_3A.6	分段 S	— — — — — — — — — — — —
	TaNAC024_3A.7	分段 S	— — — — — — — — — — — —
13	TaNAC092_2B.1	354	14—138	无 None	65.14	-0.386	31.18	6.67	叶绿体/细胞核 Ch / Nu
	TaNAC092_2B.3	235	14—136	无 None	57.87	-0.506	31.91	5.27	胞外/细胞核 Ex / Nu
	TaNAC092_2B.7	分段 S	— — — — — — — — — — — —

表3

表4

图4

图5

图6

表5

表6

小麦参考基因组（IWGSC RefSeq v1.1）注释的TaNAC转录本与小麦tae-miRNA的靶向关系预测结果"

miRNA名称 miRNA ID	期望值 Expectation	抑制方式 Inhibition	TaNAC转录本名称 TaNAC transcripts ID	靶基因长度 Length (bp)	是否可变剪切 AS or not	NAM结构域编码起止位置 NAM coding start_end (bp)	miRNA结合起止位置 Target miRNA start_end (bp)	靶基因与miRNA匹配的序列 Target aligned fragment (5′-3′)	匹配方式 Alignment
tae-miR164	1	裂解 Cleavage	TraesCS5A02G049100.1	918	是 Yes	40—423	653—673	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS5A02G049100.2	921	是 Yes	40—423	656—676	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS5B02G054200.1	921	是 Yes	40—423	656—676	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS5B02G054200.2	924	是 Yes	40—423	659—679	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS5D02G059700.1	909	是 Yes	40—423	644—664	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS5D02G059700.2	912	是 Yes	40—423	647—667	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS7A02G334800.1	1092	否 No	64—444	770—790	AGCUCGUGCCCUGCUUCUCCA	:: :::::::::::::::::
			TraesCS7A02G464100.1	924	否 No	34—435	653—673	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS7A02G464800.1	927	否 No	34—438	656—676	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS7B02G246300.1	1089	否 No	55—444	767—787	AGCUCGUGCCCUGCUUCUCCA	:: :::::::::::::::::
			TraesCS7B02G364600.1	924	否 No	34—435	653—673	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS7B02G365300.1	918	否 No	34—429	647—667	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS7D02G342300.1	1089	否 No	64—444	767—787	AGCUCGUGCCCUGCUUCUCCA	:: :::::::::::::::::
			TraesCS7D02G451700.1	939	否 No	34—438	656—676	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
			TraesCS7D02G452500.1	927	否 No	34—435	656—676	AGCAAGUGCCCUGCUUCUCCA	::: ::::::::::::::::
	1.5	裂解 Cleavage	TraesCS2A02G338300.1	1068	否 No	40—414	680—700	CGCACGUGACCUGCUUCUCCA	::::::: ::::::::::::
			TraesCS2B02G343600.1	1065	否 No	40—414	680—700	CGCACGUGACCUGCUUCUCCA	::::::: ::::::::::::
			TraesCS2D02G324700.1	1062	否 No	40—414	677—697	CGCACGUGACCUGCUUCUCCA	::::::: ::::::::::::
	2	裂解 Cleavage	TraesCS7A02G305200.1	870	否 No	34—414	665—685	AGCAAGUGUCCUGCUUCUCCG	::: :::.:::::::::::.
			TraesCS7B02G205600.1	879	否 No	34—414	674—694	AGCAAGUGUCCUGCUUCUCCG	::: :::.:::::::::::.
			TraesCS7D02G302000.1	876	否 No	34—420	671—691	AGCAAGUGUCCUGCUUCUCCG	::: :::.:::::::::::.
miRNA名称 miRNA ID	期望值 Expectation	抑制方式 Inhibition	TaNAC转录本名称 TaNAC transcripts ID	靶基因长度 Length (bp)	是否可变剪切 AS or not	NAM结构域编码起止位置 NAM coding start_end (bp)	miRNA结合起止位置 Target miRNA start_end (bp)	靶基因与miRNA匹配的序列 Target aligned fragment (5′-3′)	匹配方式 Alignment
tae-miR1128	3.5	裂解 Cleavage	TraesCS3A02G339600.1	945	是 Yes	97—480	246—266	GUUCGGGAGCAGGGAGUGGUA	:::::. : :::::::.:::
			TraesCS3A02G339600.2	882	是 Yes	97—651	371—391	GUUCGGGAGCAGGGAGUGGUA	:::::. : :::::::.:::
			TraesCS3B02G371200.1	939	否 No	85—468	234—254	GUUCGGGAGCAGGGAGUGGUA	:::::. : :::::::.:::
			TraesCS3D02G333100.1	951	否 No	97—480	246—266	GUUCGGGAGCAGGGAGUGGUA	:::::. : :::::::.:::
			TraesCS4B02G328800.1	1044	否 No	34—414	180—200	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS4B02G328900.1	1059	否 No	49—429	195—215	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS4B02G329100.1	1059	否 No	52—432	198—218	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS4D02G325800.1	1065	否 No	52—432	198—218	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS5A02G500400.1	1017	否 No	22—402	195—215	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS5A02G500500.1	1110	否 No	52—432	168—188	AAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS5A02G500600.1	1071	否 No	52—432	198—218	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
			TraesCS5A02G500700.1	2085	否 No	19—399	198—218	GAUCGGGGAGAGGGAGUGGUA	::::. ::::::::.:::
tae-miR408	3	翻译 Translation	TraesCS6A02G378500.1	936	否 No	31—441	812—832	CCCAGGGCGGCGGCAGUGCAG	::::::.: ::::::::::
	3.5	裂解 Cleavage	TraesCS4B02G320300.1	1104	否 No	25—408	622—642	UUCAAGGAGGAGGCGGAGCAG	.:: :::.:::::.: ::::
			TraesCS4D02G316800.1	1104	否 No	25—408	622—642	UUCAAGGAGGAGGCGGAGCAG	.:: :::.:::::.: ::::
			TraesCS5A02G491700.1	1047	否 No	49—429	622—642	UUCAAGGAGGAGGCGGAGCAG	.:: :::.:::::.: ::::
tae-miR9780	3	翻译 Translation	TraesCSU02G120000.1	1239	否 No	22—450	136—156	GACGCGUACGCCGCCGACCCG	: ::::: :. ::::::::::
	3.5	裂解 Cleavage	TraesCS6A02G065600.1	1227	否 No	22—444	136—156	GACGCGUACGGCGCAGAUCCG	: ::::: :.:::: ::.:::
	3.5	翻译 Translation	TraesCSU02G119900.1	1284	否 No	22—450	136—156	GACGCGUACGCCGCCGAUCCG	: ::::: :. ::::::.:::
tae-miR9676-5p	2.5	裂解 Cleavage	TraesCS5B02G480900.1	900	是 Yes	58—351	755—776	ACCUCAGCUACGAUGACAUCCA	: ::.:::::::::::::
tae-miR9676-5p	2.5	裂解 Cleavage	TraesCS5B02G480900.2	984	是 Yes	58—435	839—860	ACCUCAGCUACGAUGACAUCCA	: ::.:::::::::::::
tae-miR171a	3.5	裂解 Cleavage	TraesCS4D02G175700.1	1074	否 No	28—408	747—767	GCUGAUGGCACGGCACGAUCA	: :. ::::::::: :.::::
tae-miR444a	3.5	翻译 Translation	TraesCS7A02G349500.1	1107	否 No	31—420	1011—1031	GGAGCAACUUGGAGGCAGCAG	: :::::.: :::::::::.
tae-miR444b	3.5	翻译 Translation	TraesCS7A02G349500.1	1107	否 No	31—420	1011—1031	GGAGCAACUUGGAGGCAGCAG	: :::::.: :::::::::.
tae-miR9677a	3.5	裂解 Cleavage	TraesCS1A02G276000.1	774	否 No	13—405	296—317	UGGUCUACUCCGCCGGCGGCCA	.::::::.::..::::::

表6

表7

图7

参考文献 35

[1]	FUJITA M, FUJITA Y, MARUYAMA K, SEKI M, HIRATSU K, OHME-TAKAGI M, TRAN L S, YAMAGUCHI-SHINOZAKI K, SHINOZAKI K. A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. The Plant Journal, 2004, 39(6): 863-876. doi: 10.1111/tpj.2004.39.issue-6
[2]	TRAN L S, NAKASHIMA K, SAKUMA Y, SIMPSON S D, FUJITA Y, MARUYAMA K, FUJITA M, SEKI M, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. The Plant Cell, 2004, 16(9): 2481-2498. doi: 10.1105/tpc.104.022699
[3]	GUO Y, GAN S. AtNAP, a NAC family transcription factor, has an important role in leaf senescence. The Plant Journal, 2006, 46(4): 601-612. doi: 10.1111/tpj.2006.46.issue-4
[4]	HU H, DAI M, YAO J, XIAO B, LI X, ZHANG Q, XIONG L. Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(35): 12987-12992.
[5]	XU B, OHTANI M, YAMAGUCHI M, TOYOOKA K, WAKAZAKI M, SATO M, KUBO M, NAKANO Y, SANO R, HIWATASHI Y, MURATA T, KURATA T, YONEDA A, KATO K, HASEBE M, DEMURA T. Contribution of NAC transcription factors to plant adaptation to land. Science, 2014, 343(6178): 1505-1508. doi: 10.1126/science.1248417
[6]	LV S, GUO H, ZHANG M, WANG Q, ZHANG H, JI W. Large-scale cloning and comparative analysis of TaNAC genes in response to stripe rust and powdery mildew in wheat (Triticum aestivum L.). Genes (Basel), 2020, 11(9): 1073. doi: 10.3390/genes11091073
[7]	FENG H, DUAN X, ZHANG Q, LI X, WANG B, HUANG L, WANG X, KANG Z. The target gene of tae-miR164, a novel NAC transcription factor from the NAM subfamily, negatively regulates resistance of wheat to stripe rust. Molecular Plant Pathology, 2014, 15(3): 284-296. doi: 10.1111/mpp.2014.15.issue-3
[8]	RIECHMANN J L, HEARD J, MARTIN G, REUBER L, JIANG C, KEDDIE J, ADAM L, PINEDA O, RATCLIFFE O J, SAMAHA R R, CREELMAN R, PILGRIM M, BROUN P, ZHANG J Z, GHANDEHARI D, SHERMAN B K, YU G. Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. Science, 2000, 290(5499): 2105-2110. doi: 10.1126/science.290.5499.2105
[9]	SOUER E, VANHOUWELINGEN A, KLOOS D, MOL J, KOES R. The no apical meristem gene of petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell, 1996, 85(2): 159-170. doi: 10.1016/S0092-8674(00)81093-4
[10]	AIDA M, ISHIDA T, FUKAKI H, FUJISAWA H, TASAKA M. Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant. The Plant Cell, 1997, 9(6): 841-857. doi: 10.1105/tpc.9.6.841
[11]	OOKA H, SATOH K, DOI K, NAGATA T, OTOMO Y, MURAKAMI K, MATSUBARA K, OSATO N, KAWAI J, CARNINCI P, HAYASHIZAKI Y, SUZUKI K, KOJIMA K, TAKAHARA Y, YAMAMOTO K, KIKUCHI S. Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Research, 2003, 10(6): 239-247. doi: 10.1093/dnares/10.6.239
[12]	OLSEN A N, ERNST H A, LEGGIO L L, SKRIVER K. NAC transcription factors: Structurally distinct, functionally diverse. Trends in Plant Science, 2005, 10(2): 79-87. doi: 10.1016/j.tplants.2004.12.010
[13]	MUROZUKA E, MASSANGE-SANCHEZ J A, NIELSEN K, GREGERSEN P L, BRAUMANN I. Genome wide characterization of barley NAC transcription factors enables the identification of grain-specific transcription factors exclusive for the Poaceae family of monocotyledonous plants. PLoS ONE, 2018, 13(12): e0209769. doi: 10.1371/journal.pone.0209769
[14]	TIAN F, YANG D C, MENG Y Q, JIN J, GAO G. PlantRegMap: Charting functional regulatory maps in plants. Nucleic Acids Research, 2020, 48(D1): D1104-D1113.
[15]	GUERIN C, ROCHE J, ALLARD V, RAVEL C, MOUZEYAR S, BOUZIDI M F. Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat (T. aestivum L.). PLoS ONE, 2019, 14(3): e0213390. doi: 10.1371/journal.pone.0213390
[16]	ZHAO D, DERKX A P, LIU D C, BUCHNER P, HAWKESFORD M J. Overexpression of a NAC transcription factor delays leaf senescence and increases grain nitrogen concentration in wheat. Plant Biology (Stuttgart), 2015, 17(4): 904-913. doi: 10.1111/plb.2015.17.issue-4
[17]	HARRINGTON S A, OVEREND L E, COBO N, BORRILL P, UAUY C. Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence. BMC Plant Biology, 2019, 19(1): 407. doi: 10.1186/s12870-019-2022-5
[18]	XUE G P, BOWER N I, MCINTYRE C L, RIDING G A, KAZAN K, SHORTER R. TaNAC69 from the NAC superfamily of transcription factors is up-regulated by abiotic stresses in wheat and recognises two consensus DNA-binding sequences. Functional Plant Biology, 2006, 33(1): 43-57. doi: 10.1071/FP05161
[19]	ZHANG L, ZHANG L, XIA C, ZHAO G, JIA J, KONG X. The novel wheat transcription factor TaNAC47 enhances multiple abiotic stress tolerances in transgenic plants. Front Plant Science, 2015, 6: 1174.
[20]	PEROCHON A, KAHLA A, VRANIC M, JIA J, MALLA K B, CRAZE M, WALLINGTON E, DOOHAN F M. A wheat NAC interacts with an orphan protein and enhances resistance to Fusarium head blight disease. Plant Biotechnology Journal, 2019, 17(10): 1892-1904. doi: 10.1111/pbi.v17.10
[21]	WANG B, WEI J, SONG N, WANG N, ZHAO J, KANG Z. A novel wheat NAC transcription factor, TaNAC30, negatively regulates resistance of wheat to stripe rust. Journal of Integrative Plant Biology, 2018, 60(5): 432-443. doi: 10.1111/jipb.v60.5
[22]	PURANIK S, SAHU P P, SRIVASTAVA P S, PRASAD M. NAC proteins: Regulation and role in stress tolerance. Trends in Plant Science, 2012, 17(6): 369-381. doi: 10.1016/j.tplants.2012.02.004
[23]	AMERES S L, ZAMORE P D. Diversifying microRNA sequence and function. Nature Reviews. Molecular Cell Biology, 2013, 14(8): 475-488. doi: 10.1038/nrm3611
[24]	ZHANG H, MAO R, WANG Y, ZHANG L, WANG C, LV S, LIU X, WANG Y, JI W. Transcriptome-wide alternative splicing modulation during plant-pathogen interactions in wheat. Plant Science, 2019, 288: 110160. doi: 10.1016/j.plantsci.2019.05.023
[25]	ZHANG X M, ZHANG Q, PEI C L, LI X, HUANG X L, CHANG C Y, WANG X J, HUANG L L, KANG Z S. TaNAC2 is a negative regulator in the wheat-stripe rust fungus interaction at the early stage. Physiological and Molecular Plant Pathology, 2018, 102: 144-153. doi: 10.1016/j.pmpp.2018.02.002
[26]	WANG H-L, ZHANG Y, WANG T, YANG Q, YANG Y, LI Z, LI B, WEN X, LI W, YIN W, XIA X, GUO H, LI Z. An alternative splicing variant of PtRD26 delays leaf senescence by regulating multiple NAC transcription factors in Populus. The Plant Cell, 2021, 33: 1594-1614. doi: 10.1093/plcell/koab046
[27]	REDDY A S, MARQUEZ Y, KALYNA M, BARTA A. Complexity of the alternative splicing landscape in plants. The Plant Cell, 2013, 25(10): 3657-3683. doi: 10.1105/tpc.113.117523
[28]	XUE F, JI W, WANG C, ZHANG H, YANG B. High-density mapping and marker development for the powdery mildew resistance gene PmAS846 derived from wild emmer wheat (Triticum turgidum var. dicoccoides). Theoretical and Applied Genetics, 2012, 124(8): 1549-1560. doi: 10.1007/s00122-012-1809-7
[29]	ZHANG H, YANG Y, WANG C, LIU M, LI H, FU Y, WANG Y, NIE Y, LIU X, JI W. Large-scale transcriptome comparison reveals distinct gene activations in wheat responding to stripe rust and powdery mildew. BMC Genomics, 2014, 15(1): 898. doi: 10.1186/1471-2164-15-898
[30]	ZHANG H, MAO R, WANG Y, ZHANG L, WANG C, LV S, LIU X, WANG Y, JI W. Transcriptome-wide alternative splicing modulation during plant-pathogen interactions in wheat. Plant Science, 2019, 288: 110160. doi: 10.1016/j.plantsci.2019.05.023
[31]	SANCHEZ-MARTIN J, WIDRIG V, HERREN G, WICKER T, ZBINDEN H, GRONNIER J, SPORRI L, PRAZ C R, HEUBERGER M, KOLODZIEJ M C, ISAKSSON J, STEUERNAGEL B, KARAFIATOVA M, DOLEZEL J, ZIPFEL C, KELLER B. Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins. Nature Plants, 2021, 7(3): 327-341. doi: 10.1038/s41477-021-00869-2
[32]	GAO P, QUILICHINI T D, ZHAI C, QIN L, NILSEN K T, LI Q, SHARPE A G, KOCHIAN L V, ZOU J, REDDY A S N, WEI Y, POZNIAK C, PATTERSON N, GILLMOR C S, DATLA R, XIANG D. Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species. Plant Biotechnology Journal, 2021, 19: 1624-1643. doi: 10.1111/pbi.v19.8
[33]	KOZOMARA A, BIRGAOANU M, GRIFFITHS-JONES S. miRBase: From microRNA sequences to function. Nucleic Acids Research, 2019, 47(D1): D155-D162. doi: 10.1093/nar/gky1141
[34]	FANG Y, XIE K, XIONG L. Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. Journal of Experimental Botany, 2014, 65(8): 2119-2135. doi: 10.1093/jxb/eru072
[35]	KIM J H, WOO H R, KIM J, LIM P O, LEE I C, CHOI S H, HWANG D, NAM H G. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science, 2009, 323(5917): 1053-1057. doi: 10.1126/science.1166386

分组 Groups	TaNAC基因 TaNAC genes	正向引物序列 Forward primer sequences (5′-3′)	反向引物序列 Reversed primer sequences (5′-3′)
1	TaNAC_NTL5_7B	ATGACCCACCCTTCCTCGTC	TTACTTGCCGTAGATGCACAG
2	TaNAC008_3A	ATGGAAACTCTACGTGACATGGCT	CTACAAACTGATCCCACCACGGT
3	TaNAC013_3A	ATGACATGGTGCAATAGCTTC	TCAGGGACCAAAGCCCGTC
4	TaNAC017_5A	ATGGAGACCCCGCCGCCG	TCATGTAGAGGACTTCCATAAC
5	TaNAC011_7D	ATGGACGACGGTGCAACG	TCACCCATGATGATCCTGGT
6	TaNAC017_5D	ATGGAGACCCCGCCGCCG	CTTTGTGCATCATCTTGTCATG
7	TaNAC024_3D	ATGAACAGGGGTCACATCAGC	CTATCCATGATGATCTTGGTTGTC
8	TaNAC039_6B	ATGGACACTTTCTCCCATGTTC	CTATTTCCACAGATGAGCCGAG
9	TaNAC073_3B	ATGGACAAGGGTCACCCAGGGTAC	CTAGTACTGGTAAGGCCATTGCAC
10	TaNAC092_2A	TCCAGACGGCAAATTCCAG	TCAGTAGCCCCACGGCGC
11	TaNAC092_2D	ATGGAGCACAGCGAGCAGG	TCAGTAGCCCCACGGCGC
12	TaNAC024_3A	ATGAACAGGGGTCACATCAGC	CTATGAGATCTCCCGGTTCTTCTC
13	TaNAC092_2B	ATGGAGCACAGCGAGCAGG	AGCTTGCTTGGTCAGTAGCC

植物 Plants	miRNA前体数量 Number of miRNA precursors	成熟miRNA数量 Number of mature miRNAs	miRNA家族数量 Number of miRNA families	基因组大小 Genome sizes (G)
普通小麦 Triticum aestivum	122	125	99	14.5
玉米 Zea mays	174	325	31	2.4
水稻 Oryza sativa	604	738	342	0.5
二穗短柄草 Brachypodium distachyon	317	525	183	0.385
拟南芥 Arabidopsis thaliana	326	428	215	0.13