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作物学报 ›› 2020, Vol. 46 ›› Issue (6): 869-877.doi: 10.3724/SP.J.1006.2020.94110

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

高抗黑胫病烤烟BAC文库的构建及分析

董庆园1,4,马德清1,4,杨学2,刘勇1,黄昌军1,袁诚1,方敦煌1,于海芹1,童治军1,沈俊儒3,许银莲5,罗美中2,李永平1,曾建敏1,*()   

  • 收稿日期:2019-07-29 接受日期:2020-01-15 出版日期:2020-06-12 网络出版日期:2020-01-24
  • 通讯作者: 曾建敏
  • 作者简介:董庆园, E-mail: BlueSkies183@163.com|马德清, E-mail: 1847494695@qq.com|杨学, E-mail: 919231424@qq.com
  • 基金资助:
    中国烟草总公司云南省公司科技项目(2017YN04);中国烟草总公司云南省公司科技项目(2019530000241004)

Construction and characterization of a BAC library for flue-cured tobacco line with high resistance to blank shank

DONG Qing-Yuan1,4,MA De-Qing1,4,YANG Xue2,LIU Yong1,HUANG Chang-Jun1,YUAN Cheng1,FANG Dun-Huang1,YU Hai-Qin1,TONG Zhi-Jun1,SHEN Jun-Ru3,XU Yin-Lian5,LUO Mei-Zhong2,LI Yong-Ping1,ZENG Jian-Min1,*()   

  • Received:2019-07-29 Accepted:2020-01-15 Published:2020-06-12 Published online:2020-01-24
  • Contact: Jian-Min ZENG
  • Supported by:
    Grants of Yunnan Province Tobacco Company(2017YN04);Grants of Yunnan Province Tobacco Company(2019530000241004)

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摘要：

烟草是重要的模式植物。本研究利用pIndigoBAC536-S载体及Hind III限制性内切酶酶解烟草基因组DNA的方法, 构建了烟草新品系14-60的细菌人工染色体(BAC)文库。该文库共包含414,720个克隆, 保存在1080块384板中。随机挑选的120个烟草BAC克隆检测结果表明, 外源插入片段大小为97.0~145.5 kb, 平均约为123 kb, 空载率极低(0), 覆盖烟草基因组11倍。用烟草hemA基因、eIF4E-1基因、NtFT基因的特异引物进一步验证, 该文库质量高、可用性强, 为烟草黑胫病抗性基因的克隆以及其他重要农艺性状和品质性状等功能基因克隆研究提供了基础资源。

关键词: 烟草, 基因组DNA, 细菌人工染色体(BAC), 基因筛选

Abstract:

Tobacco (N. tabacum) is an important model crop in molecular biology research. In this study, a bacterial artificial chromosome (BAC) library of a flue-cured tobacco line 14-60 with high blank shank resistance and good quality was constructed. High molecular weight DNA was isolated using intact nuclei from tobacco, partially cleaved with Hind III and cloned into the pIndigoBAC536-S vector. The BAC library consisted of 414,720 clones arrayed in one thousand and eighty 384-microtite plates, with an average insert size of 123 kb ranging from 97.0-145.5 kb. No empty insert clone was found. Based on an estimated genome size of 4500 Mb for common tobacco, the BAC library was estimated to cover 11 times of genome equivalents. The utility of the library was further confirmed by screening the library with the primers of tobacco hemA, NtFT, and eIF4E-1 genes. The high capacity library will serve as a giant genomic resource for map-based cloning of quantitative trait loci or genes associated with important agronomic and smoking quality traits or resistance to blank shank, physical mapping and comparative genome analysis.

Key words: tobacco, genomic DNA, bacterial artificial chromosome (BAC), gene screening

图1

烟草plug DNA部分酶切 图中第1泳道为λ ladder PFG Marker, 2~9泳道分别为半块plug的酶切条带。酶浓度依次为0、0.3、0.5、1.0、1.5、2.0、4.0、10.0 U μL-1。酶切时间为15 min。"

图2

烟草基因组DNA的第1次筛选 1%琼脂糖胶脉冲场电泳条件为0.5×TBE, 1 s-50 s, 120°, 6 V cm-1, 14℃, 18 h。Marker为λ ladder PFG。"

图3

烟草基因组DNA的第2次筛选 1%琼脂糖胶脉冲场电泳条件为0.5×TBE, 14℃, 18 h, 4 s-4 s, 120°, 6 V cm-1。"

图4

洗脱产物DNA的浓度检测 λDNA标准液1 μL, 浓度分别为1、2、3、4 ng μL-1; a1、a2、b1、b2电洗脱产物各1 μL。"

表1

烟草BAC文库统计分析"

名称
Name		 指标
Index
克隆数 Total clones 414720
384孔板数 Total 384-well plates 1080
载体 BAC vector pIndigoBAC536-S
感受态细胞 Competent cell DH10B
平均插入片段大小 Average insert size (kb) 123
烟草基因组覆盖度 Tobacco genome equivalent 11×
空载率 Insert-empty BACs (%) 0

图5

烟草BAC文库随机克隆的插入片段检测(部分) 为部分随机挑选的40个克隆的插入片段检测图片。Marker为λ ladder PFG。"

图6

一级混合池2号板132~166编号的PCR筛选结果(hemA) Marker为Trans2K DNA marker, 从上到下依次是2000、1000、50、500、250、100 bp。"

图7

一级混合池2号板161~190编号的PCR筛选结果(eIF4E-1) Marker为Trans2K DNA marker, 从上到下依次是2000、1000、750、500、250、100 bp。"

图8

一级混合池9号板898~927编号PCR筛选结果(NtFT) Marker为Trans2K DNA marker, 从上到下依次是2000、1000、750、500、250、100 bp。"

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