芸薹属植物MYBL2基因的克隆及其在A、B、C基因组中的PCR鉴别

doi:10.3864/j.issn.0578-1752.2023.03.002

摘要/Abstract

摘要：

【目的】MYBL2负调控拟南芥花青素和原花青素的生物合成。从芸薹属6个物种的不同叶色材料中克隆MYBL2基因，分析其序列和表达模式，探究其在芸薹属植物花青素生物合成途径中的功能，为油菜的品质、抗逆性、观赏性等性状改良提供参考。【方法】以芸薹属6个物种19份供试材料的总DNA为模板，同源克隆MYBL2基因，并进行多序列比对和进化树分析；对白菜、甘蓝型油菜、芥菜型油菜以及埃塞俄比亚芥的紫叶材料进行遮光处理，结合转录组和qRT-PCR分析MYBL2基因表达水平；对甘蓝、甘蓝型油菜、芥菜型油菜以及埃塞俄比亚芥紫、绿叶材料进行qRT-PCR分析MYBL2基因表达水平；根据克隆MYBL2-1和MYBL2-2序列的核苷酸变异位点设计特异性引物，开发能够区分MYBL2基因组来源的PCR标记。【结果】克隆获得MYBL2-1和MYBL2-2各9个同源基因共56个拷贝。其中，BcaMYBL2-1为首次获得，BcaMYBL2-1编码区序列全长为867 bp，包含2个内含子，分别为168和102 bp，编码198个氨基酸，分子量为22.69 kD，等电点（pI）为8.72。序列比对和进化分析表明，BcaMYBL2-1来源于B基因组。芸薹属6个物种MYBL2-1和MYBL2-2同源基因中，仅BraA07.MYBL2-1、BolC06.MYBL2-1和BcaMYBL2-1在不同叶色材料中存在序列差异。经遮光处理后，紫叶材料叶色变浅，在白菜紫宝5号中，BraA07.MYBL2-1和BraA02.MYBL2-2表达量分别为未遮光部分的0.7和0.4倍；在紫叶白花甘蓝型油菜中，BnaA07.MYBL2-1、BnaC06.MYBL2-1、BnaA02.MYBL2-2和BnaC02.MYBL2-2表达量分别为未遮光部分的0.4、0.5、0.4和0.4倍；在紫叶芥中，BjuA07.MYBL2-1、BjuB03.MYBL2-1、BjuA02.MYBL2-2和BjuB05.MYBL2-2表达量分别为未遮光部分的0.4、0.3、0.4和0.2倍；在紫秆埃芥中，BcaMYBL2-1、BcaB03.MYBL2-1和BcaC03.MYBL2-2表达量分别为未遮光部分的0.3、0.4和0.5倍，而BcaB05.MYBL2-2表达量为未遮光部分的2.4倍。对比芸薹属不同叶色材料MYBL2基因表达情况，结果表明，除了羽衣甘蓝，在紫叶材料中MYBL2基因大部分同源基因的表达量均高于绿叶。在羽衣甘蓝中，绿叶羽衣甘蓝BolC06.MYBL2-1和BolC02.MYBL2-2的表达量分别为紫叶羽衣甘蓝的2.5和3.5倍；在甘蓝型油菜中，紫叶白花BnaA07.MYBL2-1、BnaC06.MYBL2-1和BnaC02.MYBL2-2的表达量分别为绿叶白花的7.5、8.6和26.0倍，而绿叶白花BnaA02.MYBL2-2的表达量为紫叶白花的13.0倍；在芥菜型油菜中，紫叶芥BjuA07.MYBL2-1、BjuB03.MYBL2-1、BjuA02.MYBL2-2和BjuB05.MYBL2-2的表达量分别为四川黄籽的8.3、11.8、23.2和14.6倍；在埃塞俄比亚芥中，紫秆埃芥BcaMYBL2-1、BcaB03.MYBL2-1的表达量分别为W-BCDH76的7.1和27.6倍，而W-BCDH76的BcaB05.MYBL2-2和BcaC03.MYBL2-2的表达量则分别为紫秆埃芥的2.8和5.0倍。依据克隆的基因序列设计出5对引物，可以有效鉴别芸薹属植物MYBL2基因的A、B、C基因组来源。【结论】芸薹属紫叶材料MYBL2基因的表达与光照密切相关，而且参与花青素生物合成的调控机制与拟南芥MYBL2负调控花青素生物合成的机制有所不同。

关键词: 芸薹属植物, MYBL2基因, 同源克隆, 基因表达, 基因组PCR鉴别

Abstract:

【Objective】In Arabidopsis, MYBL2 negatively regulates the biosynthesis of anthocyanins and proanthocyanidins. The MYBL2 genes from six Brassica species with different leaf colors were cloned. By analyzing the sequence and expression pattern of MYBL2, the function of MYBL2 in the biosynthesis of anthocyanins in Brassica species was explored. 【Method】The sequences of MYBL2 from the six Brassica species with different leaf colors were obtained using homology-based cloning method and multi sequence alignment and phylogenetic tree analysis were performed. The purple leaf materials of B. rapa, B. napus, B. juncea and B. carinata were treated with shading, and the expression level of MYBL2 gene was analyzed by transcriptome sequencing and qRT-PCR. The qRT-PCR in B. oleracea, B. napus, B. juncea and B. carinata with purple and green leaves were also performed to evaluate the expression level of MYBL2. Based on the nucleotide variation sites of the cloned MYBL2-1 and MYBL2-2 sequences, PCR markers which could distinguish the genomic origin of alleles were developed. 【Result】A total of 56 copies of 9 homologs of MYBL2-1 and MYBL2-2 were cloned from 19 samples of six species of Brassica. The BcaMYBL2-1 gene was obtained for the first time. The total length of BcaMYBL2-1 sequence was 867 bp, including two introns of 168 bp and 102 bp respectively, encoding 198 amino acids, with a molecular weight of 22.69 kD and an isoelectric point (pI) of 8.72. Sequence alignment and evolutionary analysis showed that BcaMYBL2-1 was derived from B genome. Among the MYBL2-1 and MYBL2-2 copies of six species in Brassica, only BraA07.MYBL2-1, BolC06.MYBL2-1 and BcaMYBL2-1 exhibited sequence differences in different leaf color materials. After shading treatment, the leaf color of purple leaf material becomes lighter than that of the unshaded part. In Chinese cabbage Zibao 5, the expression of BraA07.MYBL2-1 and BraA02.MYBL2-2 in the shaded part were 0.7 and 0.4 times of that in the unshaded part, respectively. In Brassica napus with purple leaves and white flowers, the expression of BnaA07.MYBL2-1, BnaC06.MYBL2-1, BnaA02.MYBL2-2 and BnaC02.MYBL2-2 in the shaded part were 0.4, 0.5, 0.4 and 0.4 times of that in the unshared part, respectively. In purple leaf mustard, the expression of BjuA07.MYBL2-1, BjuB03.MYBL2-1, BjuA02.MYBL2-2 and BjuB05.MYBL2-2 in the shaded part were 0.4, 0.3, 0.4 and 0.2 times of that in the unshared part, respectively. In B. carinata with purple leaf, the expression of BcaMYBL2-1, BcaB03.MYBL2-1 and BcaC03.MYBL2-2 in the shaded part were 0.3, 0.4 and 0.5 times of that in the unshared part, respectively. However, the expression of BcaB05.MYBL2-2 in the shaded part was 2.4 times of that in the unshared part. Comparing the expression of MYBL2 gene in different leaf color materials of Brassica, the results showed that the expression of most of the homologous genes of MYBL2 gene in purple leaf materials was higher than that in green leaf materials except kale. In kale, the expression of BolC06.MYBL2-1 and BolC02.MYBL2-2 in green kale were 2.5 and 3.5 times that in purple kale, respectively. In Brassica napus, the expression of BnaA07.MYBL2-1, BnaC06.MYBL2-1 and BnaC02.MYBL2-2 in purple leaf and white flower were 7.5, 8.6 and 26.0 times of that in green leaf and white flower, while the expression of BnaA02.MYBL2-2 in green leaf and white flower was 13.0 times of that in purple leaf and white flower. The expression levels of BjuA07.MYBL2-1, BjuB03.MYBL2-1, BjuA02.MYBL2-2 and BjuB05.MYBL2-2 in Brassica juncea were 8.3 times, 11.8 times, 23.2 times and 14.6 times of those in Sichuan yellow respectively. In B. carinata with purple leaf, BcaMYBL2-1 and BcaB03.MYBL2-1 were 7.1 and 27.6 times as much as W-BCDH76, respectively. However, BcaB05.MYBL2-2 and BcaC03.MYBL2-2 genes of W-BCDH76 were 2.8 and 5.0 times as much as those of B. carinata with purple leaf, respectively. Five pairs of primers were obtained, which can effectively identify the MYBL2 from A, B and C genomes of Brassica. 【Conclusion】After shading treatment, MYBL2 gene expression was closely related to light. The regulation mechanism of MYBL2 gene in Brassica plants involved in anthocyanin biosynthesis was different from that of Arabidopsis plants in which MYBL2 gene negatively regulated anthocyanin biosynthesis.

Key words: Brassica species, MYBL2 gene, homologous cloning, gene expression, genomic PCR identification

邹婷, 刘丽莉, 向建华, 周定港, 吴金锋, 李莓, 李宝, 张大为, 严明理. 芸薹属植物MYBL2基因的克隆及其在A、B、C基因组中的PCR鉴别[J]. 中国农业科学, 2023, 56(3): 416-429.

ZOU Ting, LIU LiLi, XIANG JianHua, ZHOU DingGang, WU JinFeng, LI Mei, LI Bao, ZHANG DaWei, YAN MingLi. Cloning of MYBL2 Gene from Brassica and Its PCR Identification in Genomes A, B and C[J]. Scientia Agricultura Sinica, 2023, 56(3): 416-429.

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材料名称 Material name	类型 Type	叶片颜色 Leaf color	基因 Gene	基因编码区核苷酸变异位点（ATG至TGA） Sites of nucleotide polymorphism (bp)
材料名称 Material name	类型 Type	叶片颜色 Leaf color	基因 Gene	57	495	540-600	646	751	842
绿叶白菜 B. rapa with green leaf	白菜 B. rapa	绿色 Green	BraA07.MYBL2-1	C	G	...	A	T	T
白菜全紫株 B. rapa with whole purple plant	白菜 B. rapa	紫色 Purple	BraA07.MYBL2-1	A	T	61	G	A	A
紫宝5号 Zibao 5	白菜 B. rapa	紫色 Purple	BraA07.MYBL2-1	C	G	...	A	T	T
四川黄籽 Sichuan Yellow	芥菜型油菜 B. juncea	绿色 Green	BjuA07.MYBL2-1	A	T	61	G	A	A
紫叶芥 Purple leaf mustard	芥菜型油菜 B. juncea	紫色 Purple	BjuA07.MYBL2-1	A	T	61	G	A	A
绿叶白花 B. napus with green leaves and white flowers	甘蓝型油菜 B. napus	绿色 Green	BnaA07.MYBL2-1	A	G	...	A	T	T
紫叶白花 B. napus with purple leaves and white flowers	甘蓝型油菜 B. napus	紫色 Purple	BnaA07.MYBL2-1	A	G	...	A	T	T
				4	18	67	217	268	395	415	519	684
W-BCDH76	埃塞俄比亚芥 B. carinata	绿色 Green	BcaMYBL2-1	C	C	G	T	A	C	A	G	T
紫秆埃芥 B. carinata with purple leaf	埃塞俄比亚芥 B. carinata	紫色 Purple	BcaMYBL2-1	A	T	A	C	G	T	G	T	C
				27	58	63	268	271	399-401	732	803	884
绿叶羽衣甘蓝 Green leaf kale	甘蓝 B. oleracea	绿色 Green	BolC06.MYBL2-1	A	A	...	C	G	TTG	A	G	T
紫叶羽衣甘蓝 Purple leaf kale	甘蓝 B. oleracea	紫色 Purple	BolC06.MYBL2-1	C	G	T	A	A	...	G	T	C