玉米非自主性转座子rDt的体细胞转座序列特征

doi:10.19586/j.2095-2341.2024.0007

生物技术进展 ›› 2024, Vol. 14 ›› Issue (2): 248-256.DOI: 10.19586/j.2095-2341.2024.0007

• 研究论文 • 上一篇

玉米非自主性转座子rDt的体细胞转座序列特征

赵官涛¹^,²(), 谭景胜¹, 朱莉¹, 李玉斌¹^,³()

^1.中国农业科学院生物技术研究所，北京 100081
^2.甘肃亚盛农业研究院有限公司，兰州 730030
^3.青岛农业大学农学院，山东青岛 266109

收稿日期:2024-01-15 接受日期:2024-02-07 出版日期:2024-03-25 发布日期:2024-04-17
通讯作者: 李玉斌
作者简介:赵官涛 E-mail: zhaoguantao@163.com；
基金资助:
国家自然科学基金面上项目(32072011);青岛农业大学高层次人才科研基金项目(665/1120002)

The Sequence Characterization of the Somatic Footprints upon the Transposition of Non-autonomous Transposon rDt in Maize

Guantao ZHAO¹^,²(), Jingsheng TAN¹, Li ZHU¹, Yubin LI¹^,³()

^1.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China
^2.Gansu Yasheng Agricultural Research Institute Co. ，Ltd. ，Lanzhou 730030，China
^3.College of Agronomy，Qingdao Agricultural University，Shandong Qingdao 266109，China

Received:2024-01-15 Accepted:2024-02-07 Online:2024-03-25 Published:2024-04-17
Contact: Yubin LI

摘要/Abstract

摘要：

Dotted/rDt是玉米遗传学中最早发现的双元转座子系统之一。为了揭示玉米中非自主性转座子rDt在其自主性转座子Dt调控下的转座遗传特性，选取了a1-rDt;Dt和a1-m1::rDt;Dt 2个rDt转座子插入突变等位基因，检测玉米籽粒紫色斑点表型差异的遗传基础，利用巢式PCR与特异性酶切相结合的方法检测并鉴定了这2种材料叶片组织中rDt体细胞转座的印迹序列类型。通过构建遗传杂交群体，统计分析各群体的后代籽粒表型以及A1野生型基因的回复突变频率。结果显示，在籽粒糊粉层紫色斑点大小均一但数目极低的a1-rDt;Dt材料中，仅检测到2种体细胞转座的印迹序列类型，其中1种是没有转座子插入前A1野生型。而在籽粒糊粉层紫色斑点大小不一、排列密集的a1-m1::rDt;Dt材料中可以检测到5种体细胞转座的印迹序列类型，其中3种类型均保持A1'回复突变基因的开放阅读框；同时，a1-m1::rDt;Dt材料中A1'的回复突变频率是a1-rDt;Dt材料的大约2.6倍。研究表明，rDt在a1插入位点体细胞转座后的修复产物序列组成相对简单，与Ac/Ds等hAT超家族转座子相似，且rDt体细胞转座产生的印迹序列类型及丰富度是两个a1基因插入突变体中A1'回复突变频率高低及玉米籽粒糊粉层表型差异的遗传基础。

关键词: 转座子, 玉米, 转座印迹, rDt

Abstract:

Dotted/rDt is one of the earliest two-element transposon systems discovered in maize genetic research. The a1-rDt；Dt and the a1-m1：：rDt；Dt are two A1 mutations with distinguishable kernel spotting phenotype， few uniform spots vs many varied-size spots. To explore the genetic characteristics of the somatic and germinal transposition of the same non-autonomous transposon rDt in either mutant line under the regulation of autonomous transposon Dt，nest-PCR amplification and restriction endonuclease digestion were applied to reveal the genetic bases for the variation in the size and number of somatic reversion spots in the aleurone by sequence analysis on the footprints at the different insertion sites of two alleles. Furthermore， segregation populations were constructed for A1' reversion frequency. The results showed that only two types of somatic footprint sequence were detected in a1-rDt；Dt line， including the exact wild-type sequence prior to any insertions. In contrast， five types of somatic footprint sequence were detected in a1-m1：：rDt；Dt line with densely populated aleurone full of various purple spots， and three of them remained in the right open reading frame of possible A1' reversion genes. Meanwhile， the reversion frequency to A1' in a1-m1：：rDt；Dt lineswas 2.6 times higher than that in a1-rDt；Dt lines. These relatively simple composition of excision footprint upon somatic transpositions of rDt at a1 locus was comparable with that of other family members from the hAT transposon superfamily such as Ac/Ds. Thus， the higher reversion frequency together with multiple reversion alleles may genetically contribute to the perceptible differences from spotting phenotype pattern in aleurone of the tow tested a1-rDt alleles.

Key words: transposable elements, maize, excision footprint, rDt

中图分类号:

赵官涛, 谭景胜, 朱莉, 李玉斌. 玉米非自主性转座子rDt的体细胞转座序列特征[J]. 生物技术进展, 2024, 14(2): 248-256.

Guantao ZHAO, Jingsheng TAN, Li ZHU, Yubin LI. The Sequence Characterization of the Somatic Footprints upon the Transposition of Non-autonomous Transposon rDt in Maize[J]. Current Biotechnology, 2024, 14(2): 248-256.

图/表 8

图1 a1-rDt和a1-m1::rDt序列结构组成及不同等位基因的籽粒表型A：a1-rDt等位基因的rDt正向插入A1基因第4外显子中，8 bp的TSD序列组成为CTCATCAC；B：a1-m1::rDt等位基因的rDt反向插入A1基因第3外显子中，8 bp的TSD序列组成为CGCCGCGT；C：基因型为a1-rDt;Dt的紫色斑点籽粒；D：基因型为a1-m1::rDt;Dt的紫色斑点籽粒；E：基因型为a1-st;Dt的无色玉米籽粒；F：基因型为A1;Dt的完全紫色玉米籽粒。A和B中粉红色方框表示外显子，咖啡色方框表示内含子，灰色方框表示3'UTR，三角形表示rDt转座子，虚线箭头表示rDt转座子插入方向，实线箭头表示引物；C、D、E和F中的标尺均为2 mm。

Fig. 1 Structure of a1-rDt and a1-m1::rDt and corresponding kernel phenotypes of variousalleles

表1 PCR扩增引物

Table 1 Primers for PCR amplification

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'
A1-8	5'-ATGAGGTAATCAAGCCGACG-3'
A1-9	5'-GACACGAAGTACATCTGCAG-3'
A1-10	5'-GCTGCACAATTAGTCTCTCG-3'
A1-11	5'-TCAGTTGAATTGATGAGGCG-3'

图2 a1-rDt的体细胞转座印迹PCR检测注：M—DL2000 DNA maker；泳道1~12为a1-rDt;Dt(Dooner)；泳道13为Mo17；泳道14为H2O。

Fig. 2 Footprint detection of rDt somatic excision (SE) products from a1-rDt

表2 a1-rDt体细胞中rDt转座印迹分析

Table 2 Footprints analysis of a1-rDt somatic excision (SE)

基因型	序列组成	变异类别
A1	5′-GCCCAGCCTCATCAC-CGCGCTGG-3′	野生型
a1-rDt	5′-GCCCAGCCTCATCAC-rDt-CTCATCACCGCGCTGG-3′	插入突变
a1-rDt-SE1(9)	5′-GCCCAGCCTCATCAC-........CGCGCTGG-3′	8+0
a1-rDt-SE2(40)	5′-GCCCAGCCTCATCA.-G-.TCATCACCGCGCTGG-3′	7+1+7(8+7)

图3 a1-m1::rDt的体细胞转座印迹PCR检测注：M—DL2000 DNA maker；泳道1~10为a1-m1::rDt;Dt(Neuffer)单株；泳道11为W22；泳道12为H2O。

Fig. 3 Footprint detection of rDt somatic excision (SE) from a1-m1::rDt

表3 a1-m1::rDt体细胞中rDt转座印迹分析

Table 3 Footprints analysis of a1-m1::rDt somatic excision(SE)

基因型	序列组成	变异类别
A1	5′-CACCGTGCGGCGCA-TCGTCT-3′	野生型
a1-m1::rDt	5′-CACCGTGCGGCGCA-rDt-GCGGCGCATCGTCT-3′	插入突变
a1-m1-SE1(20)	5′-CACCGTGCGGCGC.-.CGGCGCATCGTCT-3′	7+7(8+6)
a1-m1-SE2(10)	5′-CACCGTGCGGCGC.-GC-.CGGCGCATCGTCT-3′	7+2+7(8+8)
a1-m1-SE3(1)	5′-CACCGTGCGGCGC.-TGC-.CGGCGCATCGTCT-3′	7+3+7(8+9)
a1-m1-SE4(1)	5′-CACCGTGCGGCGC.-T-..GGCGCATCGTCT-3′	7+1+6(8+6)
a1-m1-SE5(1)	5′-CACC.............GCGCATCGTCT-3′	-2+5(8-5)

表4 不同a1-m杂交组合的A1'回复突变频率

Table 4 A1' reversion frequency analysis of different a1-m crosses and reciprocal crosses

杂交组合群体	群体粒数	A1'	回复突变频率	A1'序列组成
a1-rDt;Dt×a1-rMrh;+	135 240	16	1.19×10^-4	a1-rDt-SE1
a1-rMrh;+×a1-rDt;Dt	14 950	4	2.67×10^-4	a1-rDt-SE1
a1-m1::rDt;Dt×a1-rMrh;+	26 100	14	5.39×10^-4	a1-m1-SE1
a1-rMrh;+×a1-m1::rDt;Dt	5 600	10	1.79×10^-3	a1-m1-SE1

图4 a1-rDt体细胞转座切除位点修复模型[20]

Fig.4 Repair model of a1-rDt somatic excision sites[20]

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玉米非自主性转座子rDt的体细胞转座序列特征

The Sequence Characterization of the Somatic Footprints upon the Transposition of Non-autonomous Transposon rDt in Maize

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