CN115141842A - GmABI5基因在调控植物粒重中的应用 - Google Patents
GmABI5基因在调控植物粒重中的应用 Download PDFInfo
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Abstract
本发明提供GmABI5基因在调控植物粒重中的应用。本发明首次通过CRISPR‑Cas9系统证实基因GmABI5在调控植株粒重方面的功能，不仅为植物分子育种提供重要基因资源，也为植物高产提供有效手段。
Description
技术领域
本发明涉及植物基因工程技术和遗传育种领域，具体地说，涉及GmABI5基因在调控植物粒重中的应用。
背景技术
大豆是世界上重要的粮饲兼用作物。近年来我国大豆消费量快速增长，国产大豆自给率严重不足，进口依存度较高。百粒重是大豆育种的重要目标性状，目前已定位200多个大豆粒重相关QTL位点(https://www.soybase.org/)，但克隆的基因非常有限。Lu等(2017)利用大豆栽培品种HN44和野生大豆ZYD7为亲本构建的重组自交系(RIL)群体进行QTL定位，首次发现并克隆大豆百粒重调控基因PP2C-1，该基因是去磷酸化激活油菜素内酯BR信号通路上的转录因子，通过正向调控下游控制种粒大小的基因表达从而提高粒重(Luet al.,2017)。Wang等利用228份材料进行种子大小的QTL分析，鉴定到大豆基因GmCYP78A10是大豆种子大小和百粒重的关联基因(Wang et al.,2014)。Wang等鉴定出大豆驯化过程中的关键基因GmSWEET10a/b，它在调控大豆油分及蛋白质含量的同时，也与百粒重显著相关，但基因的调控机制尚不明确(Wang et al.,2020)。因此，有必要发掘控制大豆百粒重的新基因并解析分子机制，从而选育大豆高产新品种。
ABI5属于亮氨酸拉链转录因子bZIP家族成员。bZIP蛋白结构主要由两部分组成，分别是与DNA特异性结合的碱性氨基酸区域和参与形成同二聚体或异二聚体的亮氨酸拉链结构域(Jakoby et al.,2002)。研究发现，bZIP转录因子参与了植物生长发育，包括种子萌发和发育(Wang et al.,2020；Correa et al.,2008)、花发育(Muszynski et al.,2006)等，并且在植物抵御逆境胁迫方面也发挥重要作用，如干旱(Yang et al.,2020)、盐(Gaoet al.,2011)、低温(Liao et al.,2008)及生物胁迫(He et al.,2020)等。但在大豆中未见相关bZIP家族成员参与种子发育的报道。因此，研究大豆bZIP家族成员对大豆种子发育的调控机制具有重要意义，也为提高大豆产量提供新的基因和材料。
发明内容
本发明的目的是提供GmABI5基因在调控植物粒重中的应用。
为了实现本发明目的，第一方面，本发明提供GmABI5基因在调控植物粒重中的应用，所述调控为负调控。所述植物包括豆科植物，优选大豆。
GmABI5基因来源于大豆Willimas82，其为编码如下蛋白质(a)或(b)的基因：
(a)由SEQ ID NO:2所示的氨基酸序列组成的蛋白质；
(b)SEQ ID NO:2所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质。
GmABI5基因的核苷酸序列如SEQ ID NO:1所示，或者SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列，或者，在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或者，与上述核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。
第二方面，本发明提供一种提高大豆粒重的方法，利用基因工程手段，对大豆GmABI5基因进行改造，使得该基因功能缺失或减弱，从而提高大豆粒重(百粒重)。
进一步地，所述方法包括：以GmABI5基因为靶标，设计基于CRISPR-Cas9的sgRNA序列，将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中，转化大豆，进而获得该基因功能缺失的转基因大豆。
优选地，sgRNA作用位点位于GmABI5基因第一个外显子上。
更优选地，sgRNA作用位点的核苷酸序列为5’-TGAACATGGACGAGTTCCTCA-3’。
携带有所述目的基因的表达载体可通过使用Ti质粒、植物病毒载体、直接DNA转化、微注射、电穿孔等常规生物技术方法导入植物细胞中(Weissbach，1998，Method forPlant Molecular Biology VIII，Academy Press，New York，第411-463页；Geiserson和Corey，1998，Plant Molecular Biology，2nd Edition)。
优选采用农杆菌介导法转化大豆。
第三方面，本发明提供按照所述方法获得的转基因大豆在植物育种中的应用。
育种方法包括但不限于转基因、杂交、回交、自交或无性繁殖。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明利用我国大豆种质资源优势，发掘并克隆大豆百粒重新基因GmABI5。通过CRISPR-Cas9系统证实基因GmABI5在调控植株粒重方面的功能，筛选优异等位基因，为推进大粒高产大豆新品种的培育提供理论、基因和材料。
附图说明
图1为本发明较佳实施例中GmABI5基因的克隆。其中，a：百粒重性状的Manhadun和区间内的连锁不平衡LD图。b：GmABI5基因结构和单倍型分析图。c：不同环境单倍型间百粒重显著性分析。
图2为本发明较佳实施例中区间内基因的表达热图。其中，
图3为本发明较佳实施例中Gmabi5突变体表型(A)与PCR检测结果(B)。其中，M：2000bp DNA marker；1：空白对照(H2O)；2：WT(Williams 82)；3-5：Gmabi5突变体(携带Bar基因)。
图4为本发明较佳实施例中Gmabi5基因敲除后，突变体粒重和种子大小表型鉴定。其中，A、B分别为整个植株和种子长度的表型观察；C为3个纯合突变体的编辑类型；D-F分别为种子长度、宽度和百粒重的统计数据。**表示不同处理组之间的差异具有统计学意义，**表示P<0.01。
图5为本发明较佳实施例中GmABI5基因过表达后，转基因植株百粒重表型鉴定。其中，A为转基因株系中GmABI5的表达水平，B为过表达转基因株系百粒重的统计数据。不同字母表示不同转基因株系之间的差异具有统计学意义(P<0.05)。
具体实施方式
本发明提供一种与植物粒重相关的基因GmABI5(Glyma.19g194500)，来源于大豆Willimas82。
本发明采用如下技术方案：
结合基因型和百粒重表型数据开展GWAS，结合单倍型、基因表达谱、同源基因功能注释，采用分子生物学和比较基因组学的手段，克隆得到一个在种子特异表达的粒重相关基因GmABI5，该基因位于已报道的粒重QTL区间。GmABI5基因的克隆结果见图1。区间内基因的表达热图见图2。
利用CRISPR-Cas9系统对所述GmABI5基因进行编辑，结果发现，突变体植株的百粒重明显增加。由此可以合理推断，敲除GmABI5基因，或使所述编码基因发生沉默，或使所述编码基因低表达，可增加植物百粒重。同时可以预测，当将所述编码基因导入目的植物中，或在植物中过表达GmABI5基因，也可实现调控植株粒重的目的，且预测将减少植株的粒重。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
实施例1载体构建
利用软件CRISPRP v2.0(http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR)设计基因GmABI5的sgRNA，21bp sgRNA序列(5’-TGAACATGGACGAGTTCCTCA-3’)位于基因的第一个外显子上，利用桥式PCR将U6启动子序列、sgRNA和zCas9依次连接后，利用In-fusion构建到敲除载体pCAMBIA3301上。
1、载体酶切
1)反应体系：
2)反应条件：37℃水浴1h。利用琼脂糖凝胶电泳跑胶并回收。
2、In-fusion连接
所用试剂为Clontech 5×
HD Enzyme Premix。
将回收得到的DNA片段与酶切完回收的JRH0645载体进行连接。
1)反应体系：
In-fusion 1μL
DNA片段 2μL
载体片段 2μL
2)反应条件：50℃，30min。
3、大肠杆菌Trans10转化
1)从-80℃冰箱取出Trans10感受态细胞并置于冰上进行融化，将全部连接产物加入到含50μL感受态的1.5mL EP管中，轻弹混匀后冰上静置30min；
2)42℃水浴热激1min，然后迅速冰上静置2min；
3)向每个离心管加入500μL不含抗生素的LB培养基，置于37℃摇床，200rpm振荡培养1h；
4)取出100μL菌液均匀涂在加入卡那霉素抗生素的LB平板培养基上，37℃倒置过夜培养。
6、阳性克隆的鉴定
用枪头挑取单克隆于含10μL的水中，轻轻吹打混匀，取1μL进行菌落PCR鉴定。其余菌液加到含卡那霉素抗生素的LB液体培养基中，扩大培养用于质粒提取。
1)反应体系：
2)反应程序：
预热95℃3min；变性95℃5s，退火60℃5s，延伸68℃5s(1kb/10s)，35个循环；终延伸68℃5min。
PCR产物经电泳检测，有目的片段的克隆，把对应的扩繁菌液进行测序，获得阳性克隆，并利用公司返回质粒用于后续试验。
实施例2转基因大豆植株的获得
将构建好含有GmABI5 cDNA的质粒电击转化农杆菌后，进行大豆转化。
1、农杆菌感受态的制备与转化
1)农杆菌感受态的制备
挑取农杆菌K599、EHA105单菌落分别置于5mL含相应抗生素的LB液体培养基中，K599抗性为：100μg/mL链霉素；EHA105抗性为：100μg/mL利福平(Rif)。28℃培养过夜；取过夜培养菌液500μL接种于50mL LB含相同抗生素的液体培养基中，28℃培养到OD600约为0.8，4000rpm离心5min，倒掉上清。加入30mL 10％预冷的甘油悬浮菌体，4000rpm离心5min，倒掉上清，重复此步骤。去上清后加入3mL 10％甘油悬浮菌体，将其分装在1.5mL无菌离心管中(每管200μL)，置于-80℃冰箱备用。
2)农杆菌转化
取200μL感受态细胞冰上解冻，加入4μg质粒DNA混匀后置于电击杯中，将电击杯推入电击仪中，按pulse键5s后，向电击杯中加入的500μL YEB液体培养基，转移到1.5ml的离心管中。28℃，180rpm振荡培养2h。在超净工作台中，取出200μL菌液均匀涂布在加入卡那霉素抗生素和利福平的YEB固体培养基上，28℃倒置培养36h。
2、大豆发根检测CRISPR载体编辑效率
(1)种子消毒：挑选外表完好、饱满、光滑、圆润、无斑的大豆种子。用4mL浓盐酸和100mL NaClO反应出的氯气对豆子进行灭菌，16-18小时左右取出，在超净工作台中吹干净氯气，约为0.5-1h。
(2)种子萌发：将豆子均匀摆放在催芽培养基上，每皿10粒。温室中光照培养三天左右直至胚根2cm左右。
(3)切豆子，调菌液OD值：切子叶，取萌发3-5天的豆子，自下胚轴0.3-0.5cm处切下，将子叶一分为二切开，去除顶芽。取出摇好的菌液(K599农杆菌，OD值0.5-0.7，一般切豆子前两天小摇，前一天晚上大摇，当天上午可以用)，4000rpm离心10min，液体共培培养基(3.21g/L维生素B5+0.59g/L MES+20g/L蔗糖)重悬，使菌液OD600＝0.6，将切好的豆子中加入调好的菌液，每隔10分钟用手摇一次，共浸染30min，取出吹10min左右。
(4)共培养：在共培培养基中铺上已灭菌滤纸，然后将菌液浸染过的种子均匀地摆放在培养基中(25个/皿)，28℃暗培养3天。
(5)诱导培养：培养培3天后，胚芽长长，用无菌水和加入激素的液体诱导培养基各洗4-5次，确保将农杆菌洗净并用滤纸吸干。胚芽朝上斜插入固体诱导培养基中，放入温室中光照培养。在温室培养10-14天左右发根长出。
(6)取根检测：取不同转化载体的大豆根毛，2-3根/管，一般取3个重复，CTAB法提取DNA，进行PCR检测，测序。
(7)选取可以发生编辑(碱基缺失或增加)的位点进行大豆转化。
3、大豆转化
(1)种子灭菌：用浓盐酸和NaClO反应出的氯气对豆子进行灭菌，摇菌。
(2)种子萌发：大豆种子放在培养皿中，加入无菌水浸泡9-16h，使种子刚悬浮即可，将豆子对半切开，去掉一部分胚尖，在豆子的分生区部分划伤口，泡在无菌水中。下午取出摇好的菌液，离心(4000rpm，10min)，调菌，使菌液OD值0.4～0.6，将豆子中的无菌水倒掉，加入调好的菌液，放在摇床中摇30min(28℃，200rpm左右)，取出吹10min左右，平铺在共培培养基中暗培3天。
(3)农杆菌侵染：用手术刀切去1/2下胚轴，然后在两个子叶中间将胚轴纵向切开，去掉顶芽及侧芽。将子叶节外植体放在重悬的农杆菌菌液中(OD＝0.6)，再放入25℃培养箱中侵染0.5～4h，每隔10～20min摇晃一次使外植体与菌液充分接触。侵染完成后小心倒掉多余的农杆菌菌液，取出吹10min左右，平铺在共培培养基中26℃暗培3天。
(4)恢复培养：将长出的下胚轴切除，保留3-4mm，有伤口的一面朝上斜插入恢复培养基中，10-12个外植体/皿，放入组培室中26℃、光照16h/黑暗8h条件下培养7天。
(5)芽诱导：将长出的下胚轴切除，保留3-4mm，将外植体接种到芽诱导培养基上，5个/皿，在24℃、光照16h/黑暗8h条件下培养21天。
(6)芽伸长：在丛生芽诱导后期，淘汰污染个体，切去外植体的子叶部分，在生长点的基部作新的水平方向切口，切口朝下接种至伸长培养基中，5个/皿。在24℃、光照16h/黑暗8h条件下培养21天，若有伸长的外植体可直接进行根诱导；如果外植体还有伸长迹象更换新的芽诱导培养基继续培养21天。
(7)根诱导：当伸长芽伸长至3-4cm时，从伸长芽的基部将伸长芽与丛生芽分开，接种至生根培养基中进行根诱导。生根的周期一般为2周，待生根两周后，将培养皿上的封口膜揭开，开口炼苗3-5天。
(8)移栽：移入土中，每种一棵苗就表上豆子品种，基因名称，生根日期和土培日期。加入适量水，绿肥和缓释肥，盖上一层薄膜放在光照下，使其适应强光，3天后去膜。
实施例3 GmABI5基因敲除突变体的鉴定
为了确定转基因阳性植株，取一片鲜嫩的叶片，提取DNA，进行PCR检测。其中对于CRISPR敲除突变体，检测其Basta抗性基因。利用靶点特异引物进行PCR扩增，测序，具体结果见图3。其中，1-5分别表示WT(Williams 82)、abi5-1突变体(携带Bar基因)、abi5-2突变体(携带Bar基因)、abi5-3突变体(不携带Bar基因)和空白对照(H2O)
从图3可以看出，共获得3个纯合突变体株系abi5-1、abi5-2和abi5-3，其中abi5-3不携带Bar基因。
实施例4基因编辑材料表型鉴定
本实施例所用转基因样本数量为50株，具有统计学意义。
如图4所示，GmABI5基因敲除后，在突变体植株中出现百粒重增加的表型。其中abi5-1、abi5-2、abi5-3分别表示-37，+1和-5bp移码突变类型的纯合突变体。A、B分别为整个植株和种子长度的表型观察；C为3个纯合突变体的编辑类型；D-F分别为种子长度、宽度和百粒重的统计数据。
实施例5转基因大豆表型鉴定
本实施例所用转基因样本数量最少为10株，具有统计学意义。
如图5所示，GmABI5基因过表达后，在转基因植株中出现百粒重减少的表型。其中OE-1、OE-2、OE-3和OE-4分别表示不同的过表达转基因株系。A为转基因株系中GmABI5的表达水平，B为过表达转基因株系百粒重的统计数据。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
参考文献:
[1]Correa,L.,Riano-Pachon,D.,Schrago,C.,dos Santos,R.,Mueller-Roeber,B.,Vincentz,M.(2008).The role of bZIP transcription factors in green plantevolution:adaptive features emerging from four founder genes.PLOS ONE,3,e2944.
[2]Gao,S.,Chen,M.,Xu,Z.,Zhao,C.,Li,L.,Xu,H.,Tang,Y.,Zhao,X.,Ma,Y.(2011).The soybean GmbZIP1 transcription factor enhances multiple abioticstress tolerances in transgenic plants.PLANT MOL BIOL,75,537-553.
[3]He,Q.,Cai,H.,Bai,M.,Zhang,M.,Qin,Y.(2020).A soybean bZIPtranscription factor GmbZIP19confers multiple biotic and abiotic stressresponses in plant.INT J MOL SCI,21,4701-4720.
[4]Jakoby,M.,Weisshaar,B.,Drge-Laser,W.,Vicente-Carbajosa,J.,Tiedemann,J.,Kroj,T.,Parcy,F.(2002).bZIP transcription factors inArabidopsis.TRENDS PLANT SCI,7,106-111.
[5]Liao,Y.,Zou,H.,Wei,W.,Hao,Y.,Tian,A.,Huang,J.,Liu,Y.,Zhang,J.,Chen,S.(2008).Soybean GmbZIP44,GmbZIP62 and GmbZIP78 genes function asnegative regulator of ABA signaling and confer salt and freezing tolerance intransgenic Arabidopsis.PLANTA,228,225-240.
[6]Lu,X.,Xiong,Q.,Cheng,T.,Li,T.,Liu,X.,Bi,Y.,Li,W.,Zhang,W.,Ma,M.,Lai,Y.,Du,W.,Man,W.,Chen,S.,Zhang,J.(2017).A PP2C-1allele underlying aquantitative trait locus enhances soybean100-seed weight.MOL PLANT,10,670-684.
[7]Muszynski,M.,Dam,T.,Li,B.,Shirbroun,D.,Hou,Z.,Bruggemann,E.,Archibald,R.,Ananiev,E.,Danilevskaya.(2006).Delayed flowering1 encodes abasic leucine zipper protein that mediates floral inductive signals at theshoot apex in maize.PLANT PHYSIOL,142,1523-1536.
[8]Wang,S.,Liu,S.,Wang,J.,Yokosho,K.,Tian,Z.(2020).Simultaneouschanges in seed size,oil content,and protein content driven by selection ofSWEET homologues during soybean domestication.NATL SCI REV,7,1776-1786.
[9]Wang,X.,Li,Y.,Zhang,H.,Sun,G.,Zhang,W.,Qiu,L.(2015).Evolution andassociation analysis of GmCYP78A10 gene with seed size/weight and pod numberin soybean.MOL BIOL REP,42,489-496.
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序列表
<110> 中国农业科学院作物科学研究所
<120> GmABI5基因在调控植物粒重中的应用
<130> KHP221116268.9
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 1164
<212> DNA
<213> 大豆(Glycine max)
<400> 1
atggtgattg aggagggcga gatgaagtcc caaggtgagg tggaatcatg gctccagcaa 60
gaagcgaaga agaaccacca ttctccatta ttctcatctt cttcatatct gggaagacaa 120
acatcatcga tatactctct cacactggac gagttccagc acagtctatg cgagagcggc 180
aagaacttcg gctccatgaa catggacgag ttcctcagca gcatctggaa cgctgaagag 240
aacagccaag ccatcaccaa caacaacgtc cctctgtcgt cgaccctaac aatattaagg 300
aaacagccaa gcctccctcg ccaaccctca ctgtccctcc ctgctcctct ttgtaggaaa 360
accgttgacg aggtttggtc ccagattcaa aaagaacaaa acaagaacaa caacattagc 420
aatgttctta atgataatac cgagtctgcc cctcgccaac ccacttttgg tgagatgact 480
ttggaggatt ttttggttaa ggccggtgta gttcgggaaa caacgtgtgc accgccacta 540
ccggtgtctc actctcatca gccgcattac gcgaataata acaacaacgt tgcaatggct 600
ccttcttttg ttggtaggca tgttggtgca gtgagtaacg tggttgcacc gggttatcaa 660
caagttgttg gggaggcttc tggtgggtat ggtaagaggg atcataatgg tggtgggtat 720
cattgttttg gaggtggtgg tggtgggtat ggggtggggc ccacaatggg aatgggtggg 780
cctgtgagtc cggctaattc gtctgatggg atagggaatg atggagggca gtttgggctt 840
gacatggggg gtttgagagg gagaaagagg gtggtggatg gtcctgtgga aaaggtggtg 900
gagagaaggc agaggaggat gatcaagaat agagagtcag ctgccagatc tagagccaga 960
aaacaggcat acacagtgga attagaagca gaactaaacc agttaagaga agagaactcc 1020
caactgaaac aggcgctggc cgagctcgag aggggaagaa aacaacagtg ttttgaggaa 1080
gtgaacgtta gtgttaaaac caaagctcag aaagcaaaag agaaactgag agctttgaga 1140
aggaacatga gttgtccttt gtga 1164
<210> 2
<211> 387
<212> PRT
<213> 大豆(Glycine max)
<400> 2
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Ser Ser Ser Tyr Leu Gly Arg Gln Thr Ser Ser Ile Tyr Ser Leu Thr
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Leu Asp Glu Phe Gln His Ser Leu Cys Glu Ser Gly Lys Asn Phe Gly
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Ser Met Asn Met Asp Glu Phe Leu Ser Ser Ile Trp Asn Ala Glu Glu
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Asn Ser Gln Ala Ile Thr Asn Asn Asn Val Pro Leu Ser Ser Thr Leu
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Thr Ile Leu Arg Lys Gln Pro Ser Leu Pro Arg Gln Pro Ser Leu Ser
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Leu Pro Ala Pro Leu Cys Arg Lys Thr Val Asp Glu Val Trp Ser Gln
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Ile Gln Lys Glu Gln Asn Lys Asn Asn Asn Ile Ser Asn Val Leu Asn
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Asp Asn Thr Glu Ser Ala Pro Arg Gln Pro Thr Phe Gly Glu Met Thr
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Leu Glu Asp Phe Leu Val Lys Ala Gly Val Val Arg Glu Thr Thr Cys
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Ala Pro Pro Leu Pro Val Ser His Ser His Gln Pro His Tyr Ala Asn
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Asn Asn Asn Asn Val Ala Met Ala Pro Ser Phe Val Gly Arg His Val
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Gly Ala Val Ser Asn Val Val Ala Pro Gly Tyr Gln Gln Val Val Gly
210 215 220
Glu Ala Ser Gly Gly Tyr Gly Lys Arg Asp His Asn Gly Gly Gly Tyr
225 230 235 240
His Cys Phe Gly Gly Gly Gly Gly Gly Tyr Gly Val Gly Pro Thr Met
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Gly Met Gly Gly Pro Val Ser Pro Ala Asn Ser Ser Asp Gly Ile Gly
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Asn Asp Gly Gly Gln Phe Gly Leu Asp Met Gly Gly Leu Arg Gly Arg
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Lys Arg Val Val Asp Gly Pro Val Glu Lys Val Val Glu Arg Arg Gln
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Arg Arg Met Ile Lys Asn Arg Glu Ser Ala Ala Arg Ser Arg Ala Arg
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Lys Gln Ala Tyr Thr Val Glu Leu Glu Ala Glu Leu Asn Gln Leu Arg
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Glu Glu Asn Ser Gln Leu Lys Gln Ala Leu Ala Glu Leu Glu Arg Gly
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Arg Lys Gln Gln Cys Phe Glu Glu Val Asn Val Ser Val Lys Thr Lys
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Ala Gln Lys Ala Lys Glu Lys Leu Arg Ala Leu Arg Arg Asn Met Ser
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Claims (9)
1.GmABI5基因在调控植物粒重中的应用，其特征在于，所述调控为负调控；
GmABI5基因为编码如下蛋白质(a)或(b)的基因：
(a)由SEQ ID NO:2所示的氨基酸序列组成的蛋白质；
(b)SEQ ID NO:2所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质。
2.根据权利要求1所述的应用，其特征在于，所述植物包括豆科植物，优选大豆。
3.提高大豆粒重的方法，其特征在于，利用基因工程手段，对大豆GmABI5基因进行改造，使得该基因功能缺失或减弱，从而提高大豆粒重；
其中，GmABI5基因同权利要求1中所述。
4.根据权利要求3所述的方法，其特征在于，所述方法包括：以GmABI5基因为靶标，设计基于CRISPR-Cas9的sgRNA序列，将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中，转化大豆，进而获得该基因功能缺失的转基因大豆。
5.根据权利要求4所述的方法，其特征在于，sgRNA作用位点位于GmABI5基因第一个外显子上。
6.根据权利要求5所述的方法，其特征在于，sgRNA作用位点的核苷酸序列为5’-TGAACATGGACGAGTTCCTCA-3’。
7.根据权利要求4-6任一项所述的方法，其特征在于，采用农杆菌介导法转化大豆。
8.按照权利要求4-7任一项所述方法获得的转基因大豆在植物育种中的应用。
9.根据权利要求8所述的应用，其特征在于，育种方法包括转基因、杂交、回交、自交或无性繁殖。
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Citations (3)
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|---|---|---|---|---|
| US20090019607A1 (en) * | 2005-10-25 | 2009-01-15 | The University Of York | Transgenic plant cells expressing a transcription factor |
| US20090130710A1 (en) * | 2005-05-09 | 2009-05-21 | Allison Kermode | Enhancing vegetative protein production in transgenic plants using seed specific promoters |
| CN106188257A (zh) * | 2015-05-05 | 2016-12-07 | 中国科学院遗传与发育生物学研究所 | 大豆转录因子GmbZIP336及其编码基因在调控种子粒重中的应用 |
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| US20090130710A1 (en) * | 2005-05-09 | 2009-05-21 | Allison Kermode | Enhancing vegetative protein production in transgenic plants using seed specific promoters |
| US20090019607A1 (en) * | 2005-10-25 | 2009-01-15 | The University Of York | Transgenic plant cells expressing a transcription factor |
| CN106188257A (zh) * | 2015-05-05 | 2016-12-07 | 中国科学院遗传与发育生物学研究所 | 大豆转录因子GmbZIP336及其编码基因在调控种子粒重中的应用 |
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