玉米rMrh/Mrh双元转座系统体细胞转座特性分析

doi:10.19586/j.2095-2341.2024.0044

生物技术进展 ›› 2024, Vol. 14 ›› Issue (4): 601-609.DOI: 10.19586/j.2095-2341.2024.0044

• 研究论文 • 上一篇

玉米rMrh/Mrh双元转座系统体细胞转座特性分析

谭景胜¹(), 赵官涛¹, 朱莉¹, 李玉斌¹^,²()

^1.中国农业科学院生物技术研究所，北京 100081
^2.青岛农业大学农学院，山东青岛 266109

收稿日期:2024-03-07 接受日期:2024-04-22 出版日期:2024-07-25 发布日期:2024-08-07
通讯作者: 李玉斌
作者简介:谭景胜 E-mail: jingsheng.tan@pku-iaas.edu.cn；
基金资助:
国家自然科学基金面上项目(31871642);青岛农业大学高层次人才科研基金项目(665/1120002);中国农业科学院科技创新工程“青年英才”计划项目(2019-026)

The Sequence Characterization of Somatic Excision Footprints from Two-element Transposable System of rMrh/Mrh in Maize

Jingsheng TAN¹(), Guantao ZHAO¹, Li ZHU¹, Yubin LI¹^,²()

^1.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China
^2.College of Agronomy，Qingdao Agricultural University，Shandong Qingdao 266109，China

Received:2024-03-07 Accepted:2024-04-22 Online:2024-07-25 Published:2024-08-07
Contact: Yubin LI

摘要/Abstract

摘要：

基因组中转座子的插入和剪切都会使插入位点的序列发生改变，包括基因突变，进而导致表型变异或基因组进化。Mrh/rMrh是玉米Mutator超家族中首个经遗传学鉴定的双元转座子系统，其中仅有非自主性转座子rMrh完成了基因克隆及序列测定。通过遗传杂交后代的表型分离比确定Mrh活性系中仅有一个自主性转座子Mrh调控报告基因a1位点的rMrh发生转座。为了明确rMrh转座子在玉米体细胞中的转座剪切修复对宿主a1基因功能的影响，以及分处两个遗传位点的自主性Mrh转座子对同一a1位点rMrh剪切转座后序列修复类型的影响，对转座修复位点的PCR扩增产物进行了分子克隆及测序。结果显示，rMrh在玉米体细胞中a1位点转座后产生两种足迹类型，一种是精确修复，另一种是末端反向重复序列（terminal inverted repeat，TIR）残留。同时，在不同遗传位点的自主性转座子Mrh的调控下，rMrh在玉米体细胞中原初位点发生转座剪切时的修复类型相对单一，既能通过精确修复恢复宿主基因功能，也会因为残留碱基导致宿主基因功能的突变，但是不同遗传位点的自主性转座子Mrh的修复产物序列不尽相同。研究结果明确了经典Mrh/rMrh双元转座子系统中rMrh的转座剪切修复特性，在丰富对Mutator转座子遗传特性认识的同时，也为进一步应用这一转座子系统创制玉米新种质资源和功能基因组学遗传材料奠定了理论基础。

关键词: 双元转座系统, rMrh/Mrh, 玉米, Mutator, 体细胞转座足迹

Abstract:

Transposable element insertions and excisions are prevalent forces in driving phenotypic variations， gene mutations and genome evolution. Mrh/rMrh transposons are the components of the first genetically characterized two-element transposon system from Mutator superfamily in maize， wherein rMrh transposon， the nonautonomous component has been cloned for sequence characterization. Through phenotypic analysis on the segregation ratio of progeny segregants from genetic cross population， we verified the existence of a single copy autonomous Mrh transposon to catalyse the excision of rMrh from the reporter a1 allele in the maize genome. To elucidate the effects of somatic excision and repairing pattern of rMrh on the function of host a1 gene， and the effects of two autonomous Mrh elements from distinct genetic loci on the somatic excision and repairing pattern of rMrh element at the identical a1 locus， PCR amplification and cloning were applied to recover the simple somatic excision products for sequencing. Results suggested two types of major somatic transposition footprints upon the somatic excision of rMrh transposon in maize， one is precise excision repairing， and another is partial retentions of TIRs from rMrh. Meanwhile， under the regulation of Mrh elements from different genetic loci， the repairing patterns of rMrh somatic excision are rather simple at the primary insertion site in maize， and lead to recovery of the function of host gene through precise excision repairing or host gene mutation due to extra base-pair retentions， but the sequences of repairing products are varied between autonomous Mrh elements from different genetic loci regardless. Our results elucidated the excision repairing features of rMrh from the classic Mrh/rMrh two-element transposon system， enriched the knowledge of genetics characteristics of Mutator transposons， and provided theoretical bases for further exploration of Mrh/rMrh transposon system for generating novel maize germplasms and genetic materials for functional genomics.

Key words: two-element transposon system, rMrh/Mrh, maize, Mutator, somatic excision footprint

中图分类号:

Q291

谭景胜, 赵官涛, 朱莉, 李玉斌. 玉米rMrh/Mrh双元转座系统体细胞转座特性分析[J]. 生物技术进展, 2024, 14(4): 601-609.

Jingsheng TAN, Guantao ZHAO, Li ZHU, Yubin LI. The Sequence Characterization of Somatic Excision Footprints from Two-element Transposable System of rMrh/Mrh in Maize[J]. Current Biotechnology, 2024, 14(4): 601-609.

图/表 8

图1 a1-rMrh基因结构及rMrh插入位点示意图注：空心方框代表A1基因的非翻译区（untranslated region，UTR），黑色方框代表外显子，直线代表内含子；黑色箭头及其下标为引物名称和方向；空心的三角形代表rMrh转座子，右下方体字母为9 bp的TSD序列；EagⅠ为rMrh转座子内的限制性内切酶识别位点。

Fig. 1 The diagram of a1-rMrh gene structure and the rMrh insertion site

表1 实验所用引物

Table 1 Primers used in the experiment

引物名称	引物序列（5′→3′）
A1-1	5′-GCGAACGTTGGGAAGACGA-3′
A1-2	5′-CACCAGAGCCTCTACAGAT-3′
A1-3	5′-ATGAGCTGCACCTGCTTGAG-3′
A1-4	5′-TCAACTGAACTTCGACGACG-3′

图2 a1-rMrh分离群体的遗传图谱及表型注：F2分离群体中a1-m紫色斑点籽粒（绿色箭头）、a1-s无色籽粒（红色箭头）以及偶尔可见的A1′完全紫色籽粒（紫色箭头）。

Fig. 2 The pedigrees and phenotypes of a1-rMrh segregation population

表2 a1-rMrh/a1-rMrh；Mrh-5/+自交后代群体的表型分析

Table 2 The phenotypic analysis of a1-rMrh/a1-rMrh；Mrh-5/+ self-pollinated progenies

材料编号	斑点籽粒/个	无斑点籽粒/个	分离比	χ²	χ²_0.05,1
YC8004-3	281	95	2.96	0.014	3.841
YC8004-20	231	75	3.08	0.039
YC8004-29	135	42	3.21	0.153
YC8004-33	184	60	3.06	0.021
YC8004-37	187	62	3.01	0.001

表3 a1-rMrh/a1-rMrh；Mrh-5/+ ×a1-rMrh/a1-rMrh；+/+测交后代群体的表型分析

Table 3 The phenotypic analysis of the test cross of a1-rMrh/a1-rMrh；Mrh-5/+×a1-rMrh/a1-rMrh；+/+

材料编号	斑点籽粒/个	无斑点籽粒/个	分离比	χ²	χ²_0.05,1
YC8402-1	99	124	0.80	2.838	3.841
YC8402-34	96	114	0.84	1.554
YC8402-35	69	72	0.95	0.063
YC8402-38	117	120	0.98	0.037
YC8402-41	135	130	1.03	0.094
YC8402-43	88	68	1.29	2.607
YC8402-44	138	122	1.13	0.988

图3 不同基因型的a1等位基因的PCR验证注：M—DNA ladder Maker；1~2—a1-rMrh/a1-rMrh；Mrh-5/Mrh-5；3~4—a1-rMrh/a1-rMrh；+/+；5~6—a1-rMrh/a1-rMrh；Mrh-5/+；7—a1-rDt/ a1-rDt；+/+；8—B73。

Fig. 3 PCR validation of a1 alleles from different genotypes

图4 a1-rMrh体细胞转座产物的足迹分析注：基因型后括弧内的数字代表检测的单克隆数量；序列组成中的正体字母表示A1序列；带下划线字母表示靶位点重复序列；斜体字母为rMrh序列；圆点表示缺失序列；红色字母表示提前终止密码子；绿线框里是检测到的微同源重复序列。

Fig. 4 Footprint sequence analysis of somatic excisions from a1-rMrh

图5 a1等位基因编码的二氢黄酮醇-4-还原酶氨基酸序列组成注：下划线部分为rMrh转座子插入位点碱基序列对应编码的氨基酸残基；灰色标注部分为rDt转座子插入位点碱基序列对应编码的氨基酸残基；每条序列最后的星号代表终止密码子。

Fig. 5 Amino acid sequences of dihydroflavonol 4-reductase (DFR) coded by different a1 alleles

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玉米rMrh/Mrh双元转座系统体细胞转座特性分析

The Sequence Characterization of Somatic Excision Footprints from Two-element Transposable System of rMrh/Mrh in Maize

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