生物工程学报  2023, Vol. 39 Issue (2): 640-652
http://dx.doi.org/10.13345/j.cjb.220551
中国科学院微生物研究所、中国微生物学会主办
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文章信息

袁星, 刘金明, 郭彩华, 亢超, 张中荣, 全绍文, 牛建新
YUAN Xing, LIU Jinming, GUO Caihua, KANG Chao, ZHANG Zhongrong, QUAN Shaowen, NIU Jianxin
核桃JrGI基因克隆及表达分析
Cloning and expression analysis of JrGI gene in walnut
生物工程学报, 2023, 39(2): 640-652
Chinese Journal of Biotechnology, 2023, 39(2): 640-652
10.13345/j.cjb.220551

文章历史

Received: July 15, 2022
Accepted: September 9, 2022
Published: September 19, 2022
核桃JrGI基因克隆及表达分析
袁星 , 刘金明 , 郭彩华 , 亢超 , 张中荣 , 全绍文 , 牛建新     
石河子大学农学院 特色果蔬栽培生理与种质资源利用兵团重点实验室, 新疆 石河子 832003
摘要GI (GIGANTEA)基因是生物节律钟关键输出基因,克隆核桃JrGI基因,并分析其在不同组织及不同时间的雌花芽表达情况,旨在为研究核桃JrGI基因的功能奠定基础。采用RT-PCR (reverse transcription-polymerase chain reaction)技术从‘新新2号’叶片中克隆获得JrGI基因全长序列,对其进行生物信息学分析、烟草亚细胞定位及表达分析。结果表明,JrGI基因全长为3 516 bp,编码1 171个氨基酸,分子量为128.60 kDa,等电点(isoelectric point)为6.13,属于亲水性蛋白;系统进化分析表明JrGI蛋白与胡杨GI蛋白亲缘关系最近。烟草亚细胞定位显示JrGI蛋白位于细胞核中。对‘新新2号’雌花芽不同时间段JrGIJrCOJrFT基因进行RT-qPCR (real-time quantitative PCR)分析,结果表明在形态分化临界期的雌花芽中表达量最高,推测JrGI在此时间对雌花芽的分化起到重要作用。另外,JrGI基因在‘新新2号’各组织中均有表达,且在叶片中表达量最高,推测JrGI基因在核桃叶片发育过程中也发挥重要功能。
关键词‘新新2号’    GI基因    基因克隆    表达分析    
Cloning and expression analysis of JrGI gene in walnut
YUAN Xing , LIU Jinming , GUO Caihua , KANG Chao , ZHANG Zhongrong , QUAN Shaowen , NIU Jianxin     
Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources, College of Agriculture, Shihezi University, Shihezi 832003, Xinjiang, China
Abstract: GI (GIGANTEA) is one of the output key genes for circadian clock in the plant. The JrGI gene was cloned and its expression in different tissues was analyzed to facilitate the functional research of JrGI. RT-PCR (reverse transcription-polymerase chain reaction) was used to clone JrGI gene in present study. This gene was then analyzed by bioinformatics, subcellular localization and gene expression. The coding sequence (CDS) full length of JrGI gene was 3 516 bp, encoding 1 171 amino acids with a molecular mass of 128.60 kDa and a theoretical isoelectric point of 6.13. It was a hydrophilic protein. Phylogenetic analysis showed that JrGI of 'Xinxin 2' was highly homologous to GI of Populus euphratica. The result of subcellular localization showed that JrGI protein was located in nucleus. The JrGI, JrCO and JrFT genes in female flower buds undifferentiated and early differentiated of 'Xinxin 2' were analyzed by RT-qPCR (real-time quantitative PCR). The results showed that the expression of JrGI, JrCO and JrFT genes were the highest on morphological differentiation, implying the temporal and special regulation of JrGI in the differential process of female flower buds of'Xinxin 2'. In addition, RT-qPCR analysis showed that JrGI gene was expressed in all tissues examined, whereas the expression level in leaves was the highest. It is suggested that JrGI gene plays a key role in the development of walnut leaves.
Keywords: Juglans regia cv. xinxin No. 2    GI gene    gene clone    expression analysis    

植物通过感受昼夜长短而调控开花时间的现象称为光周期反应(photoperiod)。光周期是影响植物开花和生长发育的重要因素。光周期开花诱导途径主要由生物节律钟(circadian clock)、光受体(photoreceptors)及调节开花基因这3个组件共同构成[1-2]。只有在特定的昼夜长度下植物才能开花,而这种光周期反应需要一个内源的计时器来感受昼夜长短变化,这一计时器被称为昼夜节律钟或生物钟[3]GI (GIGANTEA)是生物节律钟输出基因,处于生物节律钟和调控开花的基因之间[2, 4]

GI是一种植物特有的蛋白,没有已知的功能结构域。GI作为开花调控因子之一,在长日照中拟南芥通过调控CO (CONSTANS)转录因子来激活FT (FLOWERING LOCUS T)基因,使其从营养生长进入生殖生长。在长日照条件下,拟南芥GI突变体会开花延迟[5],而过表达GI则会开花提前[6]。在拟南芥的叶肉和维管束中,AtGI的表达能恢复GI-2突变株的表型,且在短日照条件下AtGI能促进FT的上调表达,这表明GI在调控拟南芥开花过程中扮演重要角色[7]。在植物中,GI蛋白具有功能的保守性,如小麦、大麦和豌豆等,其中豌豆LATEBLOOMER 1蛋白与拟南芥的AtGI在开花和昼夜节律中起到相似作用[8-9]。此外,不同日照条件下,GI基因在控制植物成花的机制上存在差异。在长短日照条件下,水稻中OsGI的过表达会导致其开花的延迟,Hdl表达上调及Hd3a表达下调[10-11]。在短日照植物牵牛花(Pharbitis nil)中,GI基因的过表达会导致其开花延迟和PnFT1表达量降低,这表明PnGI可能使PnFTl表达下调来抑制其开花[12]

核桃(Juglans regia L.)属于胡桃科胡桃属的木本植物,是世界范围种植最广的树种之一。核桃营养丰富,具有较高的医疗保健和商业价值,但核桃成花晚且结实率低[13]。因此,对核桃成花的研究至关重要。GI是植物开花光周期诱导途径中的关键基因,国内外关于核桃光周期途径调控成花的研究报道较少,因此鉴定核桃GI基因并分析其功能具有重要意义。

1 材料与方法 1.1 植物材料

根据课题组前期对新疆核桃在南疆地区花器官分化的石蜡切片观察和徒手切片观察,于2021年5月31日−6月10日(此时正处于形态未分化—形态分化初期的雌花芽,其中6月6日为形态分化临界期)[14],采集新疆维吾尔自治区南疆阿克苏地区同一核桃园内的早实核桃品种‘新新2号’(Juglans regia cv. Xinxin No. 2)的叶、叶芽、雌花芽和雄花芽为试材,每2天1次,分别选取3棵各自立地条件、树龄、树体状态与栽培管理水平一致的树,于树冠外围东、西、南、北4个方向随机采样然后进行混样,将采集的样品用锡箔纸包好,迅速放入液氮中冷冻,于−80 ℃保存备用。

1.2 JrGI基因鉴定及其cDNA全长序列克隆

以拟南芥GI基因(GenBank登录号:NM_102124)为查询序列,利用BioEdit (v7.0.9.0)对‘新新2号’转录组测序数据进行本地BLASTN,其E值设定为1e–10,最终,初步筛选到的序列被上传至NCBI进行BLAST在线比对,并确定NCBI核桃的GI基因,根据其CDS (coding sequence)区设计引物。采用ESAY spin plus Plant RNA Kit (Aidlab)和TRANS公司EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix试剂盒分别进行核桃叶片RNA的提取及其反转录。以反转录获得的cDNA为模板进行聚合酶链反应(polymerase chain reaction, PCR)扩增。PCR产物用1%的琼脂糖凝胶电泳检测后回收目的基因片段,连接到pMD19-T载体(TransGen Biotech)并转化到大肠杆菌DH5α感受态细胞,选取阳性克隆菌液送西安生工生物工程股份有限公司测序。

1.3 JrGI基因的生物信息学分析

通过在线软件ExPASy protparam tool (https://web.expasy.org/protparam/)对JrGI蛋白进行理化性质分析。利用TMHMM server v2.0和NetPhos 3.1 server在线工具分别进行跨膜结构域和蛋白质磷酸化位点预测分析;通过SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/) 在线网站进行信号肽预测;利用SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)在线工具预测其二级结构;利用MEME (http://memesuite.org/index.html)在线网站预测JrGI蛋白保守基序;从NCBI网站下载不同物种的GI蛋白序列,并使用Orthovenn2H,利用MEGA-X软件采用邻接法(neighbor-joining method, NJ)构建系统发育树,设置bootstrap值为1 000,置换模型选择P-distance,其他参数选择默认。利用在线网站STRING (https://stringdb.org)对JrGI蛋白互作关系进行预测,物种来源选择模式植物拟南芥。从核桃全基因组数据库中提取核桃JrGI基因启动子区域(上游2 000 bp),利用PlantCARE网站分析JrGI基因启动子顺式作用元件的类型、数量和功能。

1.4 亚细胞位置的预测及定位

通过在线网站Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/)对JrGI蛋白进行亚细胞定位预测。利用DNMAN软件设计带酶切位点Sma I/Spe I的基因特异引物(表 1),去终止子进行PCR扩增、测序和胶回收。利用重组克隆试剂盒ClonExpress® Ⅱ One Step Cloning (Vazyme)将JrGI亚克隆至PSuper1300-GFP空载体上,构建亚细胞定位载体35S: : JrGI-GFP。采用冻融法将构建好的载体转入农杆菌GV3101,筛选、鉴定后制备渗透液,将菌液注射入4周龄大小烟草的叶片下表皮中,注射72 h后,取样通过激光共聚焦显微镜(Nikon C2-ER)进行烟草表皮细胞的亚细胞定位分析。

表 1 不同用途的引物序列 Table 1 Primers used in this study
Primer name Description Primer sequence (5′→3′)
JrGI cDNA amplification F: ATGGCTGGTTCGTGTGAGAGG
R: TCATAAGGAAATAGTACAGCCTAATTCC
18S-qRT Expression level of 18S F: GGTCAATCTTCTCGTTCCCTT
R: TCGCATTTCGCTACGTTCTT
JrGI-qRT Expression level of JrGI F: ACACGTAGCCAAGCCAATGA
R: AGACGGCTTCAGCGAGAAAA
1.5 JrGI基因的表达分析

利用ESAY spin plus Plant RNA Kit (Aidlab)提取不同组织及不同时间雌花芽的总RNA,其RNA完整性与浓度利用琼脂糖凝胶电泳与Thermo Nano Drop 2 000仪器检测;采用反转试剂盒EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen)合成cDNA第一链。利用在线软件Primer3Plus (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi)设计定量引物,并通过NCBI的Primer-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行引物特异性验证,以18S作为内参基因(表 1),利用2×S6 Universal SYBR qPCR Mix (EnzyArtisan)进行实时荧光定量PCR,所有操作均按照说明书进行。实验设3次生物学重复和3次技术重复。各基因成员相对表达量的数据分析采用2-∆∆CT[15],利用SPSS 23对结果进行单因素方差分析(ANOVA),显著性水平为P < 0.05,并用Origin作图。

2 结果与分析 2.1 核桃JrGI基因的克隆及结构分析

以‘新新2号’叶片组织提取RNA反转录得到的cDNA为模板,对JrGI基因进行PCR扩增,1%琼脂糖凝胶电泳结果如图 1所示。测序结果表明,JrGI基因的CDS全长为3 516 bp,编码1 171个氨基酸,与核桃基因库序列(GenBank登录号:LOC109011385)相似度为99.9%,将其命名为JrGI

图 1 扩增产物电泳检测结果 Fig. 1 Electrophoresis of amplified products. M:DL2000 marker;1:目的条带 M: DL2 000 marker; 1: Target band.
2.2 JrGI蛋白的理化性质

对JrGI蛋白理化性质分析发现,该蛋白由1 171个氨基酸组成;分子式为C5740H9086N1566 O1693S46;分子量为128.60 kDa;等电点值为6.13,呈酸性;含有带正电荷残基总数(Arg+Lys) 108个,带负电荷残基总数(Asp+Glu) 123个;脂肪氨基酸系数为96.53;不稳定系数为50.18;平均亲水系数为–0.027,说明该蛋白属于亲水蛋白质。此外,磷酸化作为蛋白翻译后重要修饰,JrGI蛋白共预测到22个苏氨酸位点,其中11个苏氨酸位点大于阈值(图 2)。

图 2 预测的JrGI磷酸化位点 Fig. 2 The predicted phosphorylation sites in JrGI.
2.3 JrGI蛋白结构分析

蛋白质二级结构预测发现(图 3C),JrGI蛋白由47.50% (含561个氨基酸) α螺旋、7.79% (含92个氨基酸)延伸链、4.49% (含53个氨基酸) β转角和40.22% (含475个氨基酸)无规则卷曲组成。其中α螺旋占最高比例,其次是无规则卷曲,而β转角比例最低。此外,对JrGI蛋白的信号肽(图 3A)和跨膜结构域(图 3B)预测发现,该蛋白没有信号肽和跨膜结构,属于非分泌性蛋白。

图 3 JrGI蛋白的信号肽(A)、跨膜结构域(B)及二级结构预测(C) Fig. 3 Signal peptide (A), transmembrane domain (B) and prediction of secondary structure (C) of JrGI. A:C-score为原剪切位点值(红色);S-score为信号肽值(绿色);Y-score为综合剪切位点值(C-score和S-score的几何平均数,蓝色). B:transmembrane为跨膜结构(红色);inside为膜内(蓝色);outside为膜外(紫色) A: C-score is the cleavage site score (red), S-score is the signal peptide score (green), Y-score is the combined cleavage site score (blue). B: Transmembrane (red), inside (blue), outside (purple).
2.4 JrGI蛋白保守基序及进化分析

从不同基因数据库(NCBI、TAIR、RiceDate和Phytozome v12.1)分别下载核桃、拟南芥、葡萄、水稻、胡杨的GI蛋白序列,其中核桃GI蛋白序列为拟南芥直系同源蛋白-XP_018848106.1。将其输入到MEME在线网站进行蛋白保守基序的预测。如图 4所示,共搜索获得15个保守基序,它们的宽度在37 aa−50 aa之间,分布在整个序列的不同位点。核桃JrGI氨基酸序列上的保守基序分布情况与拟南芥、葡萄、水稻和胡杨的相一致,这说明GI在不同物种间具有高的保守性,且变化程度小。

图 4 核桃与拟南芥、葡萄、水稻、胡杨GI蛋白的保守基序分析 Fig. 4 Analysis of GI conserved motifs in Juglans regia, Arabidopsis thaliana, Vitis vinifera, Oryza sativa, and Populus euphratica.

为进一步了解不同植物间GI蛋白进化关系,通过MEGA-X软件构建系统进化树(图 5)。结果表明,核桃与胡杨亲缘关系最近,其次亲缘关系较近的为葡萄,与水稻和大麦的亲缘关系较远。

图 5 JrGI与其他植物GI系统进化树 Fig. 5 Phylogenetic tree of JrGI and GI proteins from other plants.
2.5 核桃JrGI启动子与蛋白网络图分析

为进一步了解核桃JrGI基因在植物生长发育过程的作用,对该基因的启动子进行了顺式作用元件分析(图 6A)。结果发现,该基因存在3种顺式作用元件类型,其中包括胁迫响应元件(stress responsiveness, STRE)、厌氧诱导(antioxidant response element, ARE)、干旱响应(drought inducibility, MBS/MYC/MYB)等胁迫相关响应元件;脱落酸响应(abscisic acid responsiveness, ABRE)、茉莉酸甲酯响应(MeJA responsiveness, CGTCA-motif/TGACG-motif)、乙烯响应(ethephon responsiveness, ERE)等激素相关响应元件;以及生长发育相关的光响应元件(light responsiveness, Box 4/G-box/ACE/Sp1)。

图 6 JrGI启动子区顺式作用元件及JrGI蛋白相互作用网络预测 Fig. 6 Cis-acting regulatory elements and protein interaction network of JrGI. A:JrGI启动子区顺式作用元件. B:JrGI蛋白相互作用网络. 核桃同源蛋白的名称用红色标出 Cis-acting regulatory elements in promoter of JrGI (A) and protein interaction network of JrGI constructed by referring to AtGI (B). The names of walnut homologous proteins are highlighted in red.

利用STRING网站依据模式植物拟南芥的同源序列,构建了核桃JrGI蛋白相互作用关系网络。由图 6B可知,AtGI蛋白与10个蛋白存在相互作用,研究发现GIZTL (ZEITLUPE)相互作用进而调控拟南芥的昼夜节律[16-17];拟南芥中ZTL通过翻译N末端LIGHT结构域来调控GI蛋白,从而调节拟南芥的生理节律变化及其开花时间[18]GI突变会改变拟南芥的生理节律,使LHY (LATE ELONGATED HYPOCOTYL)和CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)表达量下降,而过表达GI可促进LHYCCA1的表达,但LHYCCA1也会抑制GITOC1 (TIMING OF CAB 1)的表达[19]。核桃与拟南芥的JrGI蛋白具有较高的相似性,推测可能执行与拟南芥类似的功能。

2.6 核桃JrGI蛋白的亚细胞定位

Cell-PLoc 2.0预测结果表明,JrGI定位在细胞核和细胞膜。为进一步确认JrGI在细胞中的位置,利用烟草瞬时表达技术对其进行研究。将克隆构建好的35S: : JrGI-GFP重组载体和空载体35S: : GFP (对照)转入到农杆菌GV3101,再通过注射法侵染本氏烟草叶片。瞬时表达后,在激光共聚焦显微镜下观察GFP的表达情况(图 7)。结果表明,对照组空载体的荧光信号分布烟草表皮整个细胞,而35S: : JrGI-GFP的荧光信号主要定位在细胞核中,与其功能相符。

图 7 核桃JrGI蛋白的亚细胞定位 Fig. 7 Subcellular localization analysis of walnut JrGI protein.
2.7 JrGI基因的组织及相关基因在雌花芽分化不同时间段的表达分析

为研究不同组织中JrGI基因的表达情况,以核桃叶芽的基因表达量为对照,对6月6日采集的‘新新2号’叶芽、叶、雌花芽和雄花芽样品进行qRT-PCR分析(图 8)。结果表明,JrGI在‘新新2号’叶中的表达量显著高于其他组织,推测JrGI基因在叶的生长发育过程中起到重要作用;而JrGI在‘新新2号’雌花芽和雄花芽中的表达量显著低于叶芽,表明JrGI基因在‘新新2号’中表达具有组织特异性。

图 8 JrGI基因在不同组织及相关基因在雌花芽分化不同时间段的相对表达量 Fig. 8 Relative expression of JrGI gene in different tissues and relatived genes at different time periods of female flower differentiation. Different lowercase letters indicate significant differences between treatments (P < 0.05).

为探究JrGI基因在核桃雌花芽分化过程中的作用,分析了JrGIJrCOJrFT基因分别在‘新新2号’不同时间雌花芽中的相对表达量。结果表明,以第1次采集的样品(5月31日)的表达量作为对照,6月6日采集的‘新新2号’雌花芽中JrGIJrCOJrFT的表达量显著高于其他时间。此外,6月2日采集的‘新新2号’雌花芽样品,JrGI的表达量显著高于对照,但JrCOJrFT的表达量与对照无显著差异;6月4日采集的‘新新2号’雌花芽样品JrCOJrFT的表达量显著高于对照,但JrGI的表达量与对照无显著差异。

3 讨论

在植物中,GI基因具有重要的多效性功能,如参与光合作用、气孔打开、茎伸长、光信号转导和成花诱导等许多生理和发育过程等[20]。同时,GI也是一种特异性蛋白,具有多种功能域,在花期调节、光信号、昼夜节律等过程发挥重要作用[21]。本研究成功从‘新新2号’叶片中分离克隆了GI基因,该基因编码1 171个氨基酸。二级结构预测发现,该基因α螺旋占主要组成部分,而α螺旋是一种稳定结构,能够维持蛋白的结构稳定性。磷酸化位点预测发现,该基因编码蛋白有11个苏氨酸位点大于阈值,推测其主要通过苏氨酸磷酸化来激活该蛋白质的活力,从而行使该蛋白具有的功能[22]。将JrGI蛋白与拟南芥、葡萄、水稻和胡杨中GI蛋白进行保守基序预测和多重序列比对发现,保守基序分布情况相一致,且与同是双子叶植物的葡萄和胡杨的相似度较高,这说明GI在不同物种间具高度保守性。进一步利用农杆菌将JrGI-GFP融合蛋白在烟草叶片瞬时表达证实了JrGI基因确实定位于细胞核,这与其转录调控功能吻合,与前人研究结果相一致[23]

研究发现,GI基因在拟南芥、大豆、甘薯和马铃薯中具有组织器官特异性。拟南芥中,AtGI在其叶、花、茎和根中具有较高的表达水平,且茎中的表达水平高于根[24]。在大豆中,长日照条件下,GmGI基因在叶和花芽中的表达水平最高,而短日照条件下,GmGI在根和叶中的表达水平最高[25]。甘薯中,GI基因在叶和根中的表达水平高于茎[26]。马铃薯中,StGI基因在根、匍匐茎和萼片中均具有较高的表达水平[27]。此外,在杨树中,GI-RNAi植株叶片的光合作用,气孔功能及抵抗光胁迫的能力均降低,且在长日照和中等光照条件下可诱导其叶片的衰老[28]。本研究对‘新新2号’JrGI基因在不同组织中的表达情况进行分析发现,在叶片中的表达量显著高于叶芽、雌花芽和雄花芽。表明JrGI基因在核桃叶片发育过程中发挥重要功能。

GI基因在非生物胁迫中发挥重要作用。研究表明,拟南芥中,EEL (ENHANCED EM LEVEL)与GI蛋白形成EEL-GI复合物,通过影响NCED3 (9-cis-epoxycarotenoid dioxygenase 3)的表达正向调控ABA的合成,从而提高其耐旱性[29]。干旱胁迫下,拟南芥中miRNA172E表达量依赖GI基因上调,且GI突变体对胁迫更为敏感,而过表达miRNA172的植株对胁迫的敏感度低于野生型植株[30-31]。此外,植物开花过程中,GI基因是保守的,但该基因对其开花的时间受到胁迫影响[32]。拟南芥中,GI基因正向调控其冷胁迫的耐受性,且与野生型相比,间歇冷处理对GI-3植株的开花时间影响较大,这表明AtGI可能参与调节拟南芥的开花[33]GI基因的过表达,使拟南芥开花提前,植株对盐的敏感性增强,而GI突变体则表现出更强的耐盐性和延迟开花[34]。本研究发现,在‘新新2号’JrGI基因的启动子顺式作用元件中,该基因含有干旱诱导和脱落酸响应元件,推测该基因参与调控核桃胁迫和开花过程且功能复杂。

研究发现,在长日照条件下,拟南芥中GI蛋白可与FLAVIN-BINDING、KELCH REPEAT和FKF1 (F-BOX 1)蛋白互作形成GI-FKF1复合体,CO抑制因子CDF1 (cycling doe factor1)被GI-FKF1复合体降解,从而积累开花位点T (FT)激活因子CO,促进拟南芥花的形成[35]。进一步研究表明,在长日照条件下,GI-CO-FT蛋白质处于拟南芥开花调控途径的核心位置,且FT基因通过GI和CO蛋白的调控,只能在长日照条件下转录表达,但不能在短日照条件下转录表达[36]。另外,杨树与拟南芥的开花调控途径相似,也是通过GI-CO-FT对其开花时间进行调节[37-38]。核桃必须经历花芽分化才能开花结果,而雌花芽的分化直接影响其果实产量和品质[39-40]。通过对‘新新2号’雌花芽不同时间JrGIJrCOJrFT基因的qRT-PCR分析发现,JrGIJrCOJrFT基因在6月6日采集的雌花芽样品中的表达量均显著高于对照,因此,我们推测JrGI基因可能在此时通过GI-CO-FT的途径来影响其雌花芽的分化。但对于JrGI基因是否通过GI-CO-FT的途径来调控核桃成花,需要进一步研究验证。

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核桃JrGI基因克隆及表达分析
袁星 , 刘金明 , 郭彩华 , 亢超 , 张中荣 , 全绍文 , 牛建新