欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2019, Vol. 45 ›› Issue (6): 839-847.doi: 10.3724/SP.J.1006.2019.84157

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9技术创制大豆高油酸突变系

侯智红,吴艳,程群,董利东,芦思佳,南海洋,甘卓然,刘宝辉()   

  1. 广州大学生命科学学院, 广东广州 510006
  • 收稿日期:2018-11-21 接受日期:2019-01-19 出版日期:2019-06-12 网络出版日期:2019-06-12
  • 通讯作者: 刘宝辉
  • 作者简介:E-mail: 739425367@qq.com
  • 基金资助:
    本研究由国家自然科学基金项目(31771815, 31701445, 31801384)

Creation of high oleic acid soybean mutation plants by CRISPR/Cas9

Zhi-Hong HOU,Yan WU,Qun CHENG,Li-Dong DONG,Si-Jia LU,Hai-Yang NAN,Zhuo-Ran GAN,Bao-Hui LIU()   

  1. School of Life Sciences, Guangzhou University, Guangzhou 510006, Guangdong, China
  • Received:2018-11-21 Accepted:2019-01-19 Published:2019-06-12 Published online:2019-06-12
  • Contact: Bao-Hui LIU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China.(31771815, 31701445, 31801384)

RichHTML

23

PDF

794

摘要：

大豆是重要的油料作物, 其种子脂肪酸中油酸含量是评价大豆油脂品质的重要指标之一。本研究设计了分别由AtU3d、AtU3b和AtU6-1启动子驱动、长20 bp的guide RNA (gRNA)靶点以靶向编辑GmFAD2-1A基因的外显子区, 首先将这3个靶点一起组装到pYLCRISPR/Cas9-DB载体上, 然后利用农杆菌介导的方法转化大豆材料华夏3号。通过PCR技术及测序分析对T1代转基因大豆植株靶点编辑情况进行检测, 获得纯合GmFAD2-1A大豆突变体。GmFAD2-1A突变大豆植株在株高、主茎节数、单枝分枝数、叶形、花色、种皮色、种脐色、生育期等方面与对照大豆植株没有显著差异; 而GmFAD2-1A突变体大豆种子油酸含量显著高于对照大豆品种华夏3号, 说明GmFAD2-1A是油酸代谢过程中的关键基因。本研究利用CRISPR/Cas9技术成功对控制大豆油酸基因GmFAD2-1A进行编辑, 获得稳定的纯合GmFAD2-1A大豆突变体材料, 为高油酸育种提供了新的种质资源并创建了方法。

关键词: 大豆, 脂肪酸, 油酸, GmFAD2-1A, CRISPR/Cas9

Abstract:

Oleic acid content is one of the essential indicators to evaluate quality of oil in soybean. Three sites of 20 nt guide RNA (gRNA) targeted to the exon of GmFAD2-1A were designed and transcribed from the AtU3d, AtU3b, and AtU6-1 promoters, respectively. The three target sites of gRNA were ligated to the vector pYLCRISPR/Cas9-DB, and then the recombinant plasmid was transformed into a soybean cultivar Huaxia 3 by Agrobacterium-mediated transformation. The sequences near the editing site were analyzed by the PCR method and sequencing from T1 transgenic soybean plants, homozygous GmFAD2-1A mutants were obtained using CRISPR/Cas9 technology. The agronomic traits such as plant height, main stem number, branching number per plant, leaf shape, flower color, seed coat color, hilum color and growth period were no significant difference between the transgenic soybeans and non-transformed controls. However, the content of oleic acid in the transgenic soybean seed was significantly higher than that of the control cultivar Huaxia 3, indicating that GmFAD2-1A was a key gene during synthesis of oleic acid. We succeeded in editing the GmFAD2-1A by CRISPR/Cas9 technology in soybean and obtained homozygous mutant materials, which provides new germplasm resources and method for the breeding of high oleic acid.

Key words: soybean, fatty acid, oleic acid, GmFAD2-1A, CRISPR/Cas9

图1

pYLgRNA及pYLCRISPR/Cas9-DB质粒图"

图2

3个靶点分别在GmFAD2-1A基因内的位置 左边和右边白色方框代表5' 和3' 非编码区; 黑色方框代表CDS; 实线代表内含子。"

表1

引物序列"

引物名称
Primer name		 寡核苷酸序列
Oligonucleotide sequence (5'-3')
FAD-AtU3d-T1-F1 gtcACAAAGCCACCATTCACTGT
FAD-AtU3d-T1-R1 aaacACAGTGAATGGTGGCTTTG
FAD-AtU3b-T2-F2 gtcAACCAAAATCCAAAGTTGCA
FAD-AtU3b-T2-R2 aaacTGCAACTTTGGATTTTGGT
FAD-AtU6-1-T3-F3 attGTTTGGCTGCTATGTGTTTA
FAD-AtU6-1-T3-R3 aaacTAAACACATAGCAGCCAAA
FAD2-1A-CasF-Test GAGGGATTGTAGTTCTGTTG
FAD2-1A-CasR-Test CTATGGCCCATTGGTTGCTC
U-F CTCCGTTTTACCTGTGGAATCG
gRNA-R CGGAGGAAAATTCCATCCAC
B1’ TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG
B2 AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC
B2’ TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG
B3 AGCGTGggtctcGtcttGGTCCATCCACTCCAAGCTC
B3’ TTCAGAggtctcTaagaCACTGGAATCGGCAGCAAAGG
BL AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC
Bar-F TGCCAGTTCCCGTGCTTGAA
Bar-R CTGCACCATCGTCAACCACTA

图3

pYLCRISPR/Cas9-FAD2-1A-gRNA载体构建示意图"

表2

大豆遗传转化中使用的培养基及成分"

萌发培养基
Germination
medium		 共培养培养基
Co-cultivation
medium		 诱导培养基
Shoot induction medium		 伸长培养基
Shoot elongation medium		 生根培养基
Rooting
medium
MS合成盐 MS salt mixture — — — 1× 1×
B5合成盐 B5 salt mixture 1× 1/10× 1× — —
2-(4-吗啉)乙磺酸 MES (g L-1) — 4.2 0.6 0.6 —
6-苄基腺嘌呤 6-BAP (mg L-1) — 3.2 1.6 — —
赤霉素 GA3 (mg L-1) — — — 0.5 —
羧苄青霉素Car (mg L-1) — — 50 50 —
二硫苏糖醇 DTT (mg L-1) — 150 — — —
替卡西林 Tic (mg L-1) — — 100 100 25
头孢霉素 Cef (mg L-1) — — 75 75 25
L-天冬酰胺 L-Asp (mg L-1) — — — 50 —
谷氨酰胺 Glu (mg L-1) — — — 50 —
草铵膦 Glufosinate (mg L-1) — — 250 — —
萌发培养基
Germination
medium		 共培养培养基
Co-cultivation
medium		 诱导培养基
Shoot induction
medium		 伸长培养基
Shoot elongation medium		 生根培养基
Rooting
medium
吲哚乙酸 IAA (mg L-1) — — — 0.1 —
玉米素 ZR (mg L-1) — — — 1 —
吲哚丁酸 IBA (mg L-1) — — — — 1
蔗糖 Sucrose (g L-1) 20 30 30 30 20
琼脂 Agar (g L-1) 12 6.5 8.5 9 12
pH 5.8 5.4 5.7 5.6 5.8

图4

重组质粒鉴定 A: 菌落PCR检测电泳; M: DNA marker (DM2000); 1: H2O空白对照; 2: pYLCRISPR/Cas9-DB质粒; 3~7: FAD2-1A基因敲除阳性单菌落。B: Asc I酶切鉴定pYLCRISPR/Cas9-FAD2-1A-gRNA载体; M: 1 kb DNA ladder marker; 1: pYLCRISPR/Cas9-FAD2-1A- gRNA。"

图5

转化过程及叶片涂抹草铵膦筛选结果 A: 农杆菌介导的子叶节法转化大豆; 1: 无菌苗的萌发; 2: 共培养; 3: 丛生芽的诱导; 4: 丛生芽的伸长; 5: 诱导生根; 6: 移栽土里。 B: T0转基因大豆植株草铵膦涂抹鉴定; a: 阳性转基因植株; b: 阴性转基因植株。"

图6

GmFAD2-1A突变体鉴定 A: T1代转基因大豆植株的PCR检测; M: DNA marker (DM2000); 1: H2O 空白对照; 2: H3野生型; 3~7: 转基因大豆草铵膦抗性阳性苗。B: GmFAD2-1A突变体与野生型序列比对分析; C: GmFAD2-1A突变体与野生型蛋白序列比对结果。"

表3

转基因大豆表型性状"

农艺性状
Agronomic trait		 华夏3号
Huaxia 3		 GmFAD2-1A突变体
GmFAD2-1A mutant
株高 Plant height (cm) 58.53±1.47 57.60±0.54
主茎节数 Main stem number 11±0 11±0
单株分枝数 Branching number per plant 4±0 4±0
叶形 Leaf shape 椭圆形 Oval 椭圆形 Oval
花色 Flower color 白色 White 白色 White
种皮色 Seed coat color 黄色 Yellow 黄色 Yellow
种脐色 Hilum color 浅褐色 Pale brown 浅褐色 Pale brown
生育期 Growth period (d) 107±0 107±0
蛋白 Protein (%) 40.47±0.54 40.61±0.12
油脂 Oil (%) 20.06±0.12 20.5±0.16

图7

GmFAD2-1A突变体表型及其脂肪酸组分含量 OA: 油酸; LA: 亚油酸; ALA: 亚麻酸; PA: 棕榈酸; SA: 硬脂酸。"

[1] Thelen J J, Ohlrogge J B . Metabolic engineering of fatty acid biosynthesis in plants. Metaba Eng, 2002,4:12-21.
doi: 10.1006/mben.2001.0204 pmid: 11800570
[2] 任波, 李毅 . 大豆种子脂肪酸合成代谢的研究进展. 分子植物育种, 2005,3:301-306.
Ren B, Li Y . Research advances on fatty acid biogynthesis metabolism in soybean seed. Mol Plant Breed, 2005,3:301-306 (in Chinese with English abstract).
[3] Clemente T E, Cahoon E . Soybean oil: genetic approaches for modification of functionality and total content. Plant Physiol, 2009,151:1030-1040.
[4] 邹筱, 韩粉霞, 陈明阳, 孙君明, 南金平, 闫淑荣, 杨华 . 大豆脂肪酸主要组分含量QTL定位. 作物学报, 2014,40:1595-1603.
Zou X, Han F X, Chen M Y, Sun J M, Nan J P, Yan S R, Yang H . Quantitative trait loci associated with major fatty acid components in soybean. Acta Agron Sin, 2014,40:1595-1603 (in Chinese with English abstract).
[5] 宋晓昆, 张颖君, 闫龙, 杨春燕, 郑艳艳, 蒋春志, 荆慧贤, 张孟臣, 黄占景 . 大豆脂肪酸组份相关、变异特点分析. 华北农学报, 2010,25(增刊):68-73.
Song X K, Zhang Y J, Yan L, Yang C Y, Zheng Y Y, Jiang C Z, Jing H X, Zhang M C, Huang Z J . A Study on correlation and variability of fatty acid composition contents of soybean cultivars. Acta Agric Boreali-Sin, 2010,25(suppl):68-73 (in Chinese with English abstract).
[6] Sleper D A, Shannon J G . Role of public and private soybean breeding programs in the development of soybean varieties using biotechnology. AgBioForum, 2003,6:27-32.
[7] Sleight P . Cholesterol and coronary heart disease mortality. Aust N Z J Med, 1992,22:576-579.
doi: 10.1111/j.1445-5994.1992.tb00480.x pmid: 1449442
[8] Smith G D, Song F, Sheldon T A . Cholesterol lowering and mortality: the importance of considering initial level of risk. BMJ, 1993,306:1367-1373.
doi: 10.1136/bmj.306.6893.1648 pmid: 8518602
[9] Ohlrogge J B, Kuhn D N, Stumpf P K . Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci USA, 1979,76:1194-1198.
doi: 10.1073/pnas.76.3.1194 pmid: 286305
[10] Liu Q, Brubaker C L, Green A G, Marshall D R, Sharp P J, Singh S P . Evolution of the FAD2-1 fatty acid desaturase 5' UTR intron and the molecular systematics of Gossypium(Malvaceae). Am J Bot, 2001,88:92-102.
doi: 10.2307/2657130 pmid: 11159130
[11] Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J . Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 1994,6:147-158.
doi: 10.1105/tpc.6.1.147 pmid: 7907506
[12] Zhang D, Irma L P, Stacy J P, Mongkol N, Purnima N, Sylvia W W, Robert M P, Kent D C . Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. Plant Physiol Biochem, 2009,47:462-471.
doi: 10.1016/j.plaphy.2008.12.024 pmid: 19217793
[13] Hongtrakul V, Slabaugh M, Knapp S J . A seed specific Δ 12 oleate desaturase is duplicated, rearranged, and weakly expressed in high oleic acid sunflower lines . Crop Sci, 1998,38:1245-1249.
doi: 10.2135/cropsci1998.0011183X003800050022x
[14] Li L Y, Wang X L, Gai J Y, Yu D . Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. J Plant Physiol, 2007,64:1516-1526.
doi: 10.1016/j.jplph.2006.08.007 pmid: 17141918
[15] Heppard E P, Kinney A J, Stecca K L, Miao G H . Developmental and growth temperature regulation of two different microsomalω- 6saturase genes in soybeans. Plant Physiol, 1996,110:311-319.
doi: 10.1104/pp.110.1.311 pmid: 8587990
[16] Li L Y, Wang X L, Gai J Y, Yu D Y . Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene (FAD2-1B) from soybean. DNA Seq, 2008,19:28-36.
doi: 10.1080/10425170701207208 pmid: 18300159
[17] Pham A T, Lee J D, Shannon J G, Bilyeu K D . Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait. BMC Plant Biol, 2010,10:195, doi: 10.1186/1471-2229-10-195.
doi: 10.1186/1471-2229-10-195 pmid: 20828382
[18] Pham A T, Lee J D, Shannon J G, Bilyeu K D . A novel FAD2-1A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content. Theor Appl Genet, 2011,123:793-802.
[19] Wang G L, Xu Y N . Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference. Plant Cell Rep, 2008,27:1177-1184.
doi: 10.1007/s00299-008-0535-8 pmid: 18347801
[20] Zhang L, Yang X D, Zhang Y Y, Yang J, Qi G X, Guo D Q, Xing G J, Yao Y, Xu W J, Li H Y, Li Q Y, Dong Y S . Changes in oleic acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing. Int J Genomics, 2014,2014:921-950.
doi: 10.1155/2014/921950 pmid: 4147191
[21] 杨静, 邢国杰, 牛陆, 贺红利, 杜茜, 郭东全, 袁英, 杨向东 . 反义RNA介导GmFAD2-1B基因沉默增强大豆种子中油酸的高效积累. 作物学报, 2017,43:1588-1595.
Yang J, Xing G J, Niu L, He H L, Du Q, Guo D Q, Yuan Y, Yang X D . Antisense RNA-mediated GmFAD2-1B gene silencing enhances accumulation of oleic acid in transgenic soybean seeds. Acta Agron Sin, 2017,43:1588-1595 (in Chinese with English abstract).
[22] Haun W, Coffman A, Clasen B M, Demorest Z L, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L, Voytas D F, Zhang F . Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J, 2014,12:934-940.
doi: 10.1111/pbi.12201 pmid: 24851712
[23] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, Charpentier E , A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337:816-821.
[24] Feng Z Y, Zhang B T, Ding W N, Liu X D, Yang D L, Wei P L, Cao F Q, Zhu S H, Zhang F, Mao Y F, Zhu J K . Efficient genome editing in plants using a CRISPR/Cas system. Cell Res, 2013,23:1229-1232.
doi: 10.1038/cr.2013.114 pmid: 23958582
[25] Liang Z, Zhang K, Chen K L, Gao C X . Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics, 2014,41:63-68.
doi: 10.1016/j.jgg.2013.12.001 pmid: 24576457
[26] Wang Y P, Cheng X, Shan Q W, Zhang Y, Liu J X, Gao C X, Qiu J L . Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol, 2014,32:947-951.
doi: 10.1038/nbt.2969 pmid: 25038773
[27] Shan Q W, Wang Y P, Li J, Zhang Y, Chen K L, Liang Z, Zhang K, Liu J X, Liu J X ,Jeff Xi J Z,Qiu J L, Gao C X. Targeted genome modification of crop plants using a CRISPR/Cas system. Nat Biotechnol, 2013,31:686-688.
doi: 10.1038/nbt.2650 pmid: 23929338
[28] 王加峰, 郑才敏, 刘维, 罗文龙, 王慧, 陈志强, 郭涛 . 基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建. 作物学报, 2016,42:1160-1167.
Wang J F, Zheng C M, Liu W, Luo W L, Wang H, Chen Z Q, Guo T . Construction of tgw6 mutants in rice based on CRISPR/Cas9 technology. Acta Agron Sin, 2016,42:1160-1167 (in Chinese with English abstract).
[29] Jacobs T B ,LaFayette P R,Schmitz R J,Parrott W A . Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol, 2015,15:16, doi: 10.1186/s12896-015-0131-2.
doi: 10.1186/s12896-015-0131-2 pmid: 4365529
[30] Cai Y P, Chen L, Liu X J, Chen G, Sun S, Wu C X, Jiang B J, Han T F, Hou W S . CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean. Plant Biotechnol J, 2018,16:176-185.
doi: 10.1111/pbi.12758 pmid: 28509421
[31] Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G . A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015,8:1274-1284.
doi: 10.1016/j.molp.2015.04.007 pmid: 25917172
[32] Olhoft P M, Donovan C M, Somers D A . Soybean (Glycine max) transformation using mature cotyledonary node explants. Methods Mol Biol, 2006,343:385-396.
doi: 10.1385/1-59745-130-4:385 pmid: 16988361
[33] Poirier Y, Ventre G, Caldelari D . Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium- chain-length fatty acids. Plant Physiol, 1999,121:1359-1366.
[34] Chang N W, Huang P C . Effects of the ratio of polyunsaturated and monounsaturated fatty acid to saturated fatty acid on rat plasma and liver lipid concentrations. Lipids, 1998,33:481-487.
doi: 10.1007/s11745-998-0231-9 pmid: 9625595
[35] Williams M J, Sutherland W H, Mccormick M P, De Jong S A, Walker R J, Wilkins G T . Impaired endothelial function following a meal rich in used cooking fat. J Am Coll Cardiol, 1999,33:1050-1055.
doi: 10.1016/S0735-1097(98)00681-0 pmid: 10091835
[36] Billek G . Health aspects of thermoxidized oils and fats. Eur J Lipid Sci Technol, 2000,102:587-593.
doi: 10.1002/1438-9312(200009)102:8/9<587::aid-ejlt587>3.0.co;2-#
[37] Paz M M, Martinez J C, Kalvig A B, Fonger T M, Wang K . Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep, 2006,25:206-213.
[38] Cheng T Y, Saka T ,Voqui-Dinh T H. Plant regeneration from soybean cotyledonary node segments in culture. Plant Sci Lett, 1980,19:91-99.
doi: 10.1016/0304-4211(80)90084-X
[39] Olhoft P M, Flagel L E, Donovan C M, Somers D A . Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta, 2003,216:723-735.
doi: 10.1007/s00425-002-0922-2 pmid: 12624759
[1] 陈玲玲, 李战, 刘亭萱, 谷勇哲, 宋健, 王俊, 邱丽娟. 基于783份大豆种质资源的叶柄夹角全基因组关联分析[J]. 作物学报, 2022, 48(6): 1333-1345.
[2] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[3] 王炫栋, 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花. 拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究[J]. 作物学报, 2022, 48(6): 1546-1557.
[4] 于春淼, 张勇, 王好让, 杨兴勇, 董全中, 薛红, 张明明, 李微微, 王磊, 胡凯凤, 谷勇哲, 邱丽娟. 栽培大豆×半野生大豆高密度遗传图谱构建及株高QTL定位[J]. 作物学报, 2022, 48(5): 1091-1102.
[5] 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[6] 彭西红, 陈平, 杜青, 杨雪丽, 任俊波, 郑本川, 罗凯, 谢琛, 雷鹿, 雍太文, 杨文钰. 减量施氮对带状套作大豆土壤通气环境及结瘤固氮的影响[J]. 作物学报, 2022, 48(5): 1199-1209.
[7] 王好让, 张勇, 于春淼, 董全中, 李微微, 胡凯凤, 张明明, 薛红, 杨梦平, 宋继玲, 王磊, 杨兴勇, 邱丽娟. 大豆突变体ygl2黄绿叶基因的精细定位[J]. 作物学报, 2022, 48(4): 791-800.
[8] 李瑞东, 尹阳阳, 宋雯雯, 武婷婷, 孙石, 韩天富, 徐彩龙, 吴存祥, 胡水秀. 增密对不同分枝类型大豆品种同化物积累和产量的影响[J]. 作物学报, 2022, 48(4): 942-951.
[9] 杜浩, 程玉汉, 李泰, 侯智红, 黎永力, 南海洋, 董利东, 刘宝辉, 程群. 利用Ln位点进行分子设计提高大豆单荚粒数[J]. 作物学报, 2022, 48(3): 565-571.
[10] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[11] 王娟, 张彦威, 焦铸锦, 刘盼盼, 常玮. 利用PyBSASeq算法挖掘大豆百粒重相关位点与候选基因[J]. 作物学报, 2022, 48(3): 635-643.
[12] 董衍坤, 黄定全, 高震, 陈栩. 大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能[J]. 作物学报, 2022, 48(2): 353-366.
[13] 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.
[14] 张国伟, 李凯, 李思嘉, 王晓婧, 杨长琴, 刘瑞显. 减库对大豆叶片碳代谢的影响[J]. 作物学报, 2022, 48(2): 529-537.
[15] 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711.
Viewed
Full text


Abstract

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