欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2021, Vol. 47 ›› Issue (3): 427-437.doi: 10.3724/SP.J.1006.2021.04178

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究

周冠彤(), 雷建峰, 代培红, 刘超, 李月, 刘晓东*()   

  1. 新疆农业大学农学院 / 棉花教育部工程研究中心, 新疆乌鲁木齐 830052
  • 收稿日期:2020-08-05 接受日期:2020-10-14 出版日期:2021-03-12 网络出版日期:2020-10-28
  • 通讯作者: 刘晓东
  • 作者简介:E-mail: 601930485@qq.com
  • 基金资助:
    国家自然科学基金项目(31660433);本新疆农业大学研究生科研创新项目资助(XJAUGRI2017003)

Efficient screening system of effective sgRNA for cotton CRISPR/Cas9 gene editing

ZHOU Guan-Tong(), LEI Jian-Feng, DAI Pei-Hong, LIU Chao, LI Yue, LIU Xiao-Dong*()   

  1. College of Agriculture, Xinjiang Agricultural University, Engineering Research Centre of Cotton of Ministry of Education, Urumqi 830052, Xinjiang, China
  • Received:2020-08-05 Accepted:2020-10-14 Published:2021-03-12 Published online:2020-10-28
  • Contact: LIU Xiao-Dong
  • Supported by:
    National Natural Science Foundation of China(31660433);Xinjiang Agricultural University Postgraduate Research and Innovation Project(XJAUGRI2017003)

RichHTML

17

PDF

839

摘要：

单向导RNA (sgRNA)是CRISPR/Cas9基因组编辑技术体系的重要元件之一。然而研究显示, 很多sgRNA不能有效工作, 因此需要对多个设计的候选sgRNA进行筛选, 以验证它们的有效性。早期对sgRNA有效性的验证采用的是完整编辑载体瞬时转化原生质体或者叶片的方法。这些方法费时费力, 成功率不高, 尤其是对于原生质体制备效率比较低的棉花。本研究针对GhMAPKKK2和GhAE基因分别设计靶序列, 构建了只转录sgRNA的载体: GhU6-5P::MAPKKK2-sgRNA-1300和GhU6-5P::AE-sgRNA-1300, 并通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片; 与此同时, 构建了对应完整的CRISPR/Cas9 基因组编辑载体: GhU6-5P::MAPKKK2-sgRNA-Cas9和GhU6-5P::AE-sgRNA-Cas9, 并通过农杆菌注射YZ-1野生型棉花植株的叶片。另外, 针对GhPDS、GhCLA1、GhMAPKKK2和GhAE基因分别设计靶序列并构建了GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1- sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA病毒投送载体, 通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片。以上试验均以转化对应空载体的植株为对照。对转化后的棉花叶片基因组DNA进行PCR扩增后酶切, 并对未完全消化的PCR产物进行克隆测序, 结果显示, 转化GhU6-5P::AE-sgRNA- 1300、GhU6-5P::MAPKKK2-sgRNA-Cas9、GhU6-5P::AE-sgRNA-Cas9载体的棉花植株均未检测到靶基因突变, 而转化GhU6-5P::MAPKKK2-sgRNA-1300、GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1-sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA载体的Cas9转基因阳性植株基因序列发生了改变, 突变类型包括碱基替换、碱基缺失和碱基插入。表明以Cas9转基因阳性植株为转化受体的策略可以高效真实地验证sgRNA的有效性, 排除了因转化效率低而带来的假阴性的结果, 且病毒载体投送sgRNA的策略更高效、更准确。该sgRNA高效验证体系的建立, 为棉花功能基因组学研究提供了重要的技术基础。

关键词: 棉花, 瞬时转化, CRISPR/Cas9, 基因组编辑

Abstract:

Single guide RNA (sgRNA) is one of the important elements of the CRISPR/Cas9 genome editing technology system. However, studies have shown that many sgRNAs cannot work effectively. It is worth screening to verify the effectiveness of multiple design candidate sgRNAs. Instantaneous transformation of protoplasts or leaves with complete editing vectors were used to verification of the effectiveness of sgRNA in the early stage. These methods are time-consuming and laborious, and the success rate is not high, especially for cotton with low efficiency of the protoplasmic system. In this study, target sequences were designed for GhMAPKKK2 and GhAE genes, and two vectors of GhU6-5P::MAPKKK2-sgRNA-1300, GhU6-5P::AE-sgRNA-1300 which transcibed only sgRNA were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through Agrobacterium; meanwhile, two corresponding complete CRISPR/Cas9 genome editing vectors of GhU6-5P::MAPKKK2-sgRNA-Cas9 and GhU6-5P::AE-sgRNA-Cas9 were constructed and injected YZ-1 wild-type cotton leaves with Agrobacterium. In addition, target sequences were designed for GhPDS, GhCLA1, GhMAPKKK2, and GhAE genes, respectively, and GhU6-5P-2::PDS-sgRNA- CLCrVA, GhU6-5P-2::CLA1-sgRNA-CLCrVA, GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-CLCrVA virus delivery vectors were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through Agrobacterium. In the above experiments, the plants transformed with the empty vector were used as controls. The genomic DNA of the transformed cotton leaves was subjected to PCR and enzyme digestion, and the PCR products which were not completely digested were cloned and sequenced. The results showed that no mutation in target gene was detected in the cotton plants transformed with the GhU6-5P::AE-sgRNA-1300, GhU6-5P::MAPKKK2-sgRNA-Cas9 and GhU6-5P::AE-sgRNA-Cas9, and the target genes mutation in the Cas9 transgenic plants transformed with GhU6-5P::MAPKKK2-sgRNA-1300, GhU6-5P-2::PDS-sgRNA-CLCrVA, GhU6-5P-2::CLA1-sgRNA-CLCrVA, GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-CLCrVA vector was uncovered. The types of mutations included base substitution, base deletion and base insertion. The results indicated that the strategy of using Cas9 transgenic plants as transformation recipients can efficiently and truly verify the effectiveness of sgRNA, which eliminated false negative results due to low transformation efficiency, and the strategy of using virus as vectors to deliver sgRNA was more efficient and accurate. The establishment of this sgRNA high-efficiency verification system provides an important technical basis for cotton functional genomics research.

Key words: cotton, transient transformation, CRISPR/Cas9, genome editing

表1

本研究中使用的引物序列"

引物名称
Primer name		 上游引物序列
Forward sequence (5'-3')		 下游引物序列
Reverse sequence (5'-3')
GhU6-5P-MAPKKK2-sg AAGGGTTCCCAGCTGACATA TATGTCAGCTGGGAACCCTT
GhU6-5P-AE-sg GAGTTTGGAGGGCTTACAAT ATTGTAAGCCCTCCAAACTC
GhU6-5P-2-PDS-sg GAAGCGAGAGATGTTCTAGG CCTAGAACATCTCTCGCTTC
GhU6-5P-2-CLA1-sg TATGCTCGCGGAATGATCAG CTGATCATTCCGCGAGCATA
Test MAPKKK2-sg CCATGTCGTAGCTTATAAAGG TCATTTACCTTCTCTTCCCAG
Test AE-sg AGACTTGTTTCAATGGACTC AATAAGCTGACAGCAGTTGG
Test PDS-sg TGCATGATCCATCACTCAAGTTT GAACGAAAGGCCCTTTCTTTC
Test CLA1-sg GGATCTGAAAGGTGAAAGGAATC TACCGTGATACTTGTCAGCAGCT

图1

基因编辑载体的酶切鉴定 A: 1、2分别为GhU6-5P::MAPKKK2-sgRNA-1300和GhU6-5P::MAPKKK2-sgRNA-Cas9. B: 1、2分别为GhU6-5P::AE-sgRNA-1300和GhU6-5P::AE-sgRNA-Cas9. C: 1~4分别为GhU6-5P-2::PDS-sgRNA-ClCrVA、GhU6-5P-2::CLA1-sgRNA-ClCrVA GhU6-5P-2::MAPKKK2- sgRNA-ClCrVA和GhU6-5P-2::AE-sgRNA-ClCrVA. M: 2K plus II DNA marker。"

图2

GhU6-5P::MAPKKK2-sgRNA靶向突变的检测 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~6为转化GhU6-5P::MAPKKK2-sgRNA-Cas9单株样品基因组PCR扩增产物酶切后结果。B: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~6为转化GhU6-5P::MAPKKK2-sgRNA-1300单株样品基因组PCR扩增产物酶切后结果。C: GhMAPKKK2-sgRNA靶序列及突变PCR产物的测序。M1发生在D亚组的GhMAPKKK2, M2、M3发生在A亚组GhMAPKKK2。D: 突变PCR产物克隆的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。"

图3

GhU6-5P::AE-sgRNA靶向突变的检测 A: M: 2K plus II DNA marker; 12、11分别为对照载体基因组PCR扩增产物酶切前后结果; 3~12为转化GhU6-5P::AE-sgRNA-Cas9单株样品基因组PCR扩增产物酶切后结果。B: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~12为转化GhU6-5P::AE-sgRNA-1300单株样品基因组PCR扩增产物酶切后结果。PCR/RE表示PCR扩增产物酶切分析。"

图4

GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果。B: M: 2K plus II DNA marker; 3~6为转化GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: M: 2K plus II DNA marker; 1~10为单克隆菌液PCR产物酶切后结果。PCR/RE表示PCR扩增产物酶切分析。"

图5

GhU6-5P-2::AE-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果。B: M: 2K plus II DNA marker; 3~7为转化GhU6-5P-2::AE-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: M: 2K plus II DNA marker; 1~10为单克隆菌液PCR产物酶切后结果。PCR/RE表示PCR扩增产物酶切分析。"

图6

GhU6-5P-2::PDS-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切后和酶切前结果。B: M: 2K plus II DNA marker; 3~5为转化GhU6-5P-2::PDS-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: GhPDS-sgRNA靶序列及突变PCR产物的测序。M1、M2发生在A亚组的GhPDS, M3发生在D亚组的GhPDS。D: 突变PCR产物的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。"

图7

GhU6-5P-2::CLA1-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果。B: M: 2K plus II DNA marker; 3~5为转化GhU6-5P-2::CLA1-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: GhCLA1-sgRNA靶序列及突变PCR产物的测序。M1, M3发生在D亚组的GhCLA1, M2发生在A亚组的GhCLA1。D: 突变PCR产物的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。"

[1] 许宗弘. 棉花枯黄萎病研究现状及展望. 知识经济, 2010,16:132.
Xu Z H. Research status and prospect of cotton Fusarium Wilt. Knowledge Econ, 2010,16:132 (in Chinese with English abstract).
[2] 任爱霞. 棉花枯黄萎病抗性遗传及生化机理研究. 浙江大学硕士学位论文, 浙江杭州, 2002.
Ren A X. Study on Inheritance and Biochemical Mechanism of Cotton Fusarium Wilt Resistance. MS Thesis of Zhejiang University, Hangzhou, Zhejiang, China, 2002 (in Chinese with English abstract).
[3] 徐立华. 我国棉花高产、高效栽培技术研究现状与发展思路. 中国棉花, 2001, (3):5-8.
Xu L H. Research status and development ideas of cotton high-yield and high-efficiency cultivation technology in my country. China Cotton, 2001, (3):5-8 (in Chinese with English abstract).
[4] 孙学振, 施培, 周治国. 我国棉花高产栽培技术理论研究现状与展望. 中国棉花, 1999, (4):2-7.
Sun X Z, Shi P, Zhou Z G. Current status and prospects of the theoretical research on cotton high-yield cultivation techniques in my country. China Cotton, 1999, (4):2-7 (in Chinese with English abstract).
[5] Sun Y, Li J, Xia L. Precise genome modification via sequence specific nucleases-mediated gene targetingfor crop improvement. Front Plant Sci, 2016,7:1928.
doi: 10.3389/fpls.2016.01928 pmid: 28066481
[6] 刘蓓, 尉玮, 王丽华. 基因编辑新技术研究进展. 亚热带农业研究, 2013,9(4):262-269.
Liu B, Wei W, Wang L H. Research progress of new technology of gene editing. Subtrop Agric Res, 2013,9(4):262-269 (in Chinese with English abstract).
[7] Cao H X, Wang W, Le H T, Vu G T. The power of CRISPR-Cas9-induced genome editing to speed up plant breeding. Int J Genomics, 2016,2016:5078796.
[8] Gilbert L A, Larson M H, Morsut L, Liu Za, Brar G A, Torres S E, Stern-Ginossar N, Brandman O, Whitehead E H, Doudna J A, Lim W A, Weissman J S, Qi L S. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013,154:442-451.
[9] Hsu P D, Lander E S, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014,157:1262-1278.
pmid: 24906146
[10] Bassett A R, Tibbit C, Ponting C P, Liu J L. Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system. Cell Rep, 2014,6:1178-1179.
[11] Barrangou R, Marraffini L A. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell, 2014,54:234-244.
[12] Mao Y F, Zhang Z J, Feng Z Y, Wei P L, Zhang H, Botella J R, Zhu J K. Development of germline specific CRISPR/Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol J, 2016,14:519-532.
doi: 10.1111/pbi.12468 pmid: 26360626
[13] Kim H, Kim S T, Ryu J, Choi M K, Kweon J, Kang B C, Ahn H M, Bae S, Kim J, Kim J S, Kim S G. A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR/Cas system. J Integr Plant Biol, 2016,58:705-712.
doi: 10.1111/jipb.12474
[14] Gao S L, Tong Y Y, Wen Z Q, Zhu L, Ge M, Chen D J, Jiang Y, Yang S. Multiplex gene editing of theYarrowia lipolytica genome using the CRISPR/Cas9 system. J Ind Microbiol Biotechnol, 2016,43:1085-1093.
pmid: 27349768
[15] Zhang F, Maeder M L, Unger-Wallace E, Hoshaw J P, Reyon D, Christian M, Li X H, Pierick C J, Dobbs D, Peterson T, Joung J K, Voytas D F. High frequency targeted mutagenesis inArabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci USA, 2010,107:12028-12033.
doi: 10.1073/pnas.0914991107 pmid: 20508152
[16] Shukla V K, Doyon Y, Miller J C, DeKelver R C, Moehle E A, Worden S E, Mitchell J C, Arnold N L, Gopalan S, Meng X D, Choi V M, Rock J M, Wu Y Y, Katibah G E, Gao Z F, McCaskill D, Simpson M A, Blakeslee B, Greenwalt S A, Butler H J, Hinkley S J, Zhang L, Rebar E J, Gregory P D, Urnov F D. Precise genome modification in the crop speciesZea mays using zinc-finger nucleases. Nature, 2009,459:437-441.
doi: 10.1038/nature07992 pmid: 19404259
[17] Townsend J A, Wright D A, Winfrey R J, Fu F L, Maeder M L, Joung J K, Voytas D F. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature, 2009,459:442-445.
pmid: 19404258
[18] 谢小东, 高军平, 李泽锋, 张剑锋, 魏攀, 罗朝鹏, 王晨, 武明珠, 翟妞, 杨军. CRISPR/Cas9介导烟草多基因编辑体系的应用. 中国烟草学报, 2019,25(4):72-80.
Xie X D, Gao J P, Li Z F, Zhang J F, Wei P, Luo Z P, Wang C, Wu M Z, Zhai N, Yang J. Application of CRISPR/Cas9 mediated tobacco multi-gene editing system. Acta Tab Sin, 2019,25(4):72-80 (in Chinese with English abstract).
[19] 王海明, 张立强, 李娜, 刘建丰, 马崇烈. 利用CRISPR/Cas9基因编辑技术敲除水稻NRR基因促进根系生长的研究. 杂交水稻, 2019,34(5):39-45.
Wang H M, Zhang L Q, Li N, Liu J F, Ma C L. Using CRISPR/Cas9 gene editing technology to knock out rice NRR gene to promote root growth. Hybrid Rice, 2019,34(5):39-45 (in Chinese with English abstract).
[20] 陈修贵. CRISPR/Cas9系统介导的棉花GhCLA1和GhVP基因编辑的研究. 华中农业大学博士学位论文, 湖北武汉, 2017.
Chen X G. Study on Cotton GhCLA1 and GhVP Gene Editing Mediated by CRISPR/Cas9 System. PhD Dissertation of Huazhong Agricultural University, Wuhan, Hubei, China, 2017 (in Chinese with English abstract).
[21] 王艳玲, 孟志刚, 李妍妍, 孟钊红, 王远, 孙国清, 朱涛, 梁成真, 蔡永萍, 郭三堆, 张锐, 林毅. CRISPR/Cas9编辑棉花精氨酸酶基因促进侧根形成和发育. 中国科学: 生命科学, 2017,47:1200-1203.
Wang Y L, Meng Z G, Li Y Y, Meng Z H, Wang Y, Sun G Q, Zhu T, Liang C Z, Cai Y P, Guo S D, Zhang R, Lin Y. CRISPR/Cas9 editing cotton arginase gene promotes lateral root formation and development. Sci Sin (Vitae), 2017,47:1200-1203 (in Chinese with English abstract).
[22] Farboud B, Meyer B J. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design. Genetics, 2015,199:959-971.
doi: 10.1534/genetics.115.175166 pmid: 25695951
[23] Chen X G, Lu X K, Shu N, Wang S, Wang J J, Wang D L, Guo L X, Ye W W. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Sci Rep, 2017,7:44304.
pmid: 28287154
[24] Gao W, Long L, Tian X Q, Xu F C, Liu J, Prashant K S, Jose R B, Song C P. Genome editing in cotton with the CRISPR/Cas9 system. Front Plant Sci, 2017,8:1364.
[25] Hu J C, Li S, Li Z L, Li H Y, Song W B, Zhao H M, Lai J S, Xia L Q, Li D W, Zhang Y L. A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize. Mol Plant Pathol, 2019,20:1463-1474.
pmid: 31273916
[26] Yin K Q, Han T, Liu G, Chen T Y, Wang Y, Yu A Y L, Liu Y L. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing. Sci Rep, 2015,5:14926.
pmid: 26450012
[27] Ali Z, Abul-Faraj A, Li L X, Ghosh N, Piatek M, Mahjoub A, Aouida M, Piatek A, Baltes N J, Voytas D F, Dinesh-Kumar S, Mahfouz M M. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant, 2015,8:1288-1291.
doi: 10.1016/j.molp.2015.02.011 pmid: 25749112
[28] Ali Z, Eid A, Ali S, Mahfouz M M. Pea early-browning virus- mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis. Virus Res, 2018,244:333-337.
pmid: 29051052
[29] Cody W B, Scholthof H B, Mirkov T E. Multiplexed gene editing and protein overexpression using aTobacco mosaic virus viral vector. Plant Physiol, 2017,175:23-35.
doi: 10.1104/pp.17.00411 pmid: 28663331
[30] Jiang N, Zhang C, Liu J Y, Guo Z H, Zhang Z Y, Han C G, Wang Y. Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing. Plant Biotechnol J, 2019,17:1302-1315.
pmid: 30565826
[31] Gu Z H, Huang C J, Li F F, Zhou X P. A versatile system for functional analysis of genes and microRNAs in cotton. Plant Biotechnol J, 2014,12:638-649.
pmid: 24521483
[32] 雷建峰, 伍娟, 陈晓俊, 於添平, 倪志勇, 李月, 张巨松, 刘晓东. 棉花花粉中高效转录U6启动子的克隆及功能分析. 中国农业科学, 2015,48:3794-3802.
Lei J F, Wu J, Chen X J, Yu T P, Ni Z Y, Li Y, Zhang J S, Liu X D. Cloning and functional analysis of the highly efficient transcription U6 promoter in cotton pollen. Sci Agric Sin, 2015,48:3794-3802 (in Chinese with English abstract).
[33] Zhu S H, Yu X L, Li Y J, Sun Y Q, Zhu Q H, Sun J. Highly efficient targeted gene editing in upland cotton using the CRISPR/Cas9 system. Int J Mol Sci, 2018,19:3000.
[34] Gao W, Long L, Tian X Q, Xu F C, Liu J, Prashant K S, Jose R B, Song C P. Genome editing in cotton with the CRISPR/Cas9 system. Front Plant Sci, 2017,8:1364.
[35] 李妮娜, 丁林云, 张志远, 郭旺珍. 棉花叶肉原生质体分离及目标基因瞬时表达体系的建立. 作物学报, 2014,40:231-239.
Li N N, Ding L Y, Zhang Z Y, Guo W Z. Isolation of mesophyll protoplast and establishment of gene transient expression system in cotton. Acta Agron Sin, 2014,40:231-239 (in Chinese with English abstract).
[1] 周静远, 孔祥强, 张艳军, 李雪源, 张冬梅, 董合忠. 基于种子萌发出苗过程中弯钩建成和下胚轴生长的棉花出苗壮苗机制与技术[J]. 作物学报, 2022, 48(5): 1051-1058.
[2] 孙思敏, 韩贝, 陈林, 孙伟男, 张献龙, 杨细燕. 棉花苗期根系分型及根系性状的关联分析[J]. 作物学报, 2022, 48(5): 1081-1090.
[3] 闫晓宇, 郭文君, 秦都林, 王双磊, 聂军军, 赵娜, 祁杰, 宋宪亮, 毛丽丽, 孙学振. 滨海盐碱地棉花秸秆还田和深松对棉花干物质积累、养分吸收及产量的影响[J]. 作物学报, 2022, 48(5): 1235-1247.
[4] 郑曙峰, 刘小玲, 王维, 徐道青, 阚画春, 陈敏, 李淑英. 论两熟制棉花绿色化轻简化机械化栽培[J]. 作物学报, 2022, 48(3): 541-552.
[5] 张艳波, 王袁, 冯甘雨, 段慧蓉, 刘海英. 棉籽油分和3种主要脂肪酸含量QTL分析[J]. 作物学报, 2022, 48(2): 380-395.
[6] 张特, 王蜜蜂, 赵强. 滴施缩节胺与氮肥对棉花生长发育及产量的影响[J]. 作物学报, 2022, 48(2): 396-409.
[7] 赵文青, 徐文正, 杨锍琰, 刘玉, 周治国, 王友华. 棉花叶片响应高温的差异与夜间淀粉降解密切相关[J]. 作物学报, 2021, 47(9): 1680-1689.
[8] 岳丹丹, 韩贝, Abid Ullah, 张献龙, 杨细燕. 干旱条件下棉花根际真菌多样性分析[J]. 作物学报, 2021, 47(9): 1806-1815.
[9] 张旺, 冼俊霖, 孙超, 王春明, 石丽, 于为常. CRISPR/Cas9编辑花生FAD2基因研究[J]. 作物学报, 2021, 47(8): 1481-1490.
[10] 曾紫君, 曾钰, 闫磊, 程锦, 姜存仓. 低硼及高硼胁迫对棉花幼苗生长与脯氨酸代谢的影响[J]. 作物学报, 2021, 47(8): 1616-1623.
[11] 马欢欢, 方启迪, 丁元昊, 池华斌, 张献龙, 闵玲. 棉花GhMADS7基因正调控棉花花瓣发育[J]. 作物学报, 2021, 47(5): 814-826.
[12] 许乃银, 赵素琴, 张芳, 付小琼, 杨晓妮, 乔银桃, 孙世贤. 基于GYT双标图对西北内陆棉区国审棉花品种的分类评价[J]. 作物学报, 2021, 47(4): 660-671.
[13] 卢合全, 唐薇, 罗振, 孔祥强, 李振怀, 徐士振, 辛承松. 商品有机肥替代部分化肥对连作棉田土壤养分、棉花生长发育及产量的影响[J]. 作物学报, 2021, 47(12): 2511-2521.
[14] 王晔, 刘钊, 肖爽, 李芳军, 吴霞, 王保民, 田晓莉. 转PSAG12-IPT基因对棉花叶片衰老及产量和纤维品质的影响[J]. 作物学报, 2021, 47(11): 2111-2120.
[15] 杨琴莉, 杨多凤, 丁林云, 赵汀, 张军, 梅欢, 黄楚珺, 高阳, 叶莉, 高梦涛, 严孙艺, 张天真, 胡艳. 棉花花器官突变体的鉴定及候选基因的克隆[J]. 作物学报, 2021, 47(10): 1854-1862.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2