CN109161553B - 一种梨转录因子PbBP及其应用 - Google Patents
一种梨转录因子PbBP及其应用 Download PDFInfo
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- CN109161553B CN109161553B CN201811148204.4A CN201811148204A CN109161553B CN 109161553 B CN109161553 B CN 109161553B CN 201811148204 A CN201811148204 A CN 201811148204A CN 109161553 B CN109161553 B CN 109161553B
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Abstract
本发明公开了一种梨木质素合成调控PbBP基因,所述梨木质素合成调控PbBP基因具有如SEQ ID NO.1所示的核苷酸序列,命名为PbBP,所述PbBP基因编码的氨基酸序列如SEQ ID NO.2所示,核苷酸长度为1194bp,氨基酸长度为397个氨基酸。调控基因PbBP及其编码蛋白应用在植物中合成木质素基因表达方面。本发明PbBP基因具有负调控作用,可以直接通过抑制植物木质素合成基因的表达从而阻碍其木质素的生物合成,减缓细胞壁发育,降低木质化程度,以提高梨果肉品质。
Description
技术领域
本发明属于植物分子生物学和基因工程技术领域,具体涉及一种梨转录因子PbBP及其应用。
背景技术
梨属于蔷薇科(Rosaceae)梨亚科(Pomaeeae)梨属(Pyrus L.)植物,是世界上重要的栽培果树之一。其中原产安徽省砀山县的砀山酥梨属白梨(Pyrus bretschneideri)系,是我国栽培面积最大的梨品种。随着水果品种的丰富和消费者生活水平的提高,砀山酥梨石细胞团含量较高且体积较大的固有缺陷日益凸显。石细胞含量的高低、石细胞团的大小以及密度不但会影响梨果实的质地和口感,而且会影响梨果实的糖含量。较高的石细胞含量会导致果实的口感风味变差,营养成分受损,从而影响了梨的营养价值和经济效益。
前人的研究发现,发育成熟的梨石细胞中含有约40%的木质素。在梨开花后的15天左右,一些果肉薄壁细胞会较早地开始发生次生壁加厚的现象,形成了石细胞原基细胞。随后,这些原基细胞周围的薄壁细胞也逐渐发生次生壁加厚。在次生壁加厚的过程中,伴随有大量的木质素单体的合成与沉积。最终木质素与纤维素微纤丝共同组成石细胞细胞壁,次生壁持续沉积增厚直至填充整个细胞内腔,大量石细胞聚集成簇形成石细胞团。这些实验观察结果表明,木质素在细胞壁上的沉积和次生细胞壁的加厚是石细胞发育的关键步骤。因此通过调控梨木质素代谢及细胞壁发育将有效改变果实中石细胞的含量。研究砀山酥梨中与木质素合成、次生壁发育相关的转录因子,可以同时调控多个结构基因,达到多点调控的效果,对于揭示梨石细胞的发育机理,为降低果实石细胞含量提高梨内在品质奠定基础。此外,木质素是构成次生壁的组成成分之一,沉积在木质部外围和茎的微管细胞中。木质素的含量高低与水果(杏、石榴、葡萄等)的果核大小及硬度、农作物抗倒伏能力以及秸秆降解再利用均密切相关,因此发掘调控木质素合成的转录因子对于改良作物关键农艺性状等具有重要意义。
KNOTTED1类似的同源盒基因—KNOX(KNOTTED1-like homeobox genes,KNOX),是维管植物中调控顶端分生组织活性的关键因子,不仅参与调控细胞的分化,而且在生物的形态建成中也发挥着重要的作用。1991年Vollbrecht.E等在玉米突变体中首次发现了同源盒基因KNOTTED1(KN1),自此KNOX表达机理的研究为解析植物发育过程揭开了新的篇章。随后在小立碗藓(Physcomitrella patens)、柳枝稷(Panicum virgatum)、拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、杨树(Populus trichocarpa)等物种中获得大量同源基因,这些基因形成了KNOX基因家族。在陆生植物中,KNOX基因家族的成员较多,并且KNOX家族成员可以划分成I型和II型两类:I类主要包括KNAT1(BREVIPEDICELLUS,BP)、KNAT2(KN1-like2)、KNAT6和STM(SHOOTMERISTEMLESS);II类主要包括KNAT3、KNAT4、KNAT5和KNAT7。该基因家族结构保守且分布广泛,几乎存在于所有植物中,但是目前为止在梨中还未见相关报道,对于梨中调控木质素合成的转录因子研究也鲜有报道。
发明内容
本发明的目的正是为了解决上述问题,而提出一种梨转录因子PbBP,该基因具有负调控作用,可以直接通过抑制植物木质素合成基因的表达从而阻碍其木质素的生物合成,导致细胞壁发育减缓且木质化程度降低。
本发明提供了一种梨转录因子PbBP,所述梨转录因子PbBP具有如SEQ ID NO.1所示的核苷酸序列,命名为PbBP,所述PbBP基因编码的氨基酸序列如SEQ ID NO.2所示,核苷酸长度为1194bp,氨基酸长度为397个氨基酸。
作为进一步地优选手段,所述基因PbBP的克隆步骤如下:提取梨总RNA并反转录成cDNA,根据公布的梨基因组序列设计特异性扩增引物PbBP-F和PbBP-R,以cDNA为模板进行PCR扩增;
所述特异性扩增引物PbBP-F和PbBP-R为:
SEQ ID NO.3:PbBP-F:5’-ATGGAAGACTACAACAGTCAAATGGATCATG-3’
SEQ ID NO.4:PbBP-R:5’-TCATGGCCCGAGACGGTAGTGAACG-3’。
一种梨转录因子PbBP在调控植物木质素合成及细胞壁发育中的应用。
一种植物超量表达载体,所述植物超量表达载体包括所述的梨转录因子PbBP构建的pMD-18T-PbBP质粒。
作为进一步地优选手段,所述植物超量表达载体采用pMD-18T-PbBP质粒和pCambia1304质粒进行双酶切构建植物表达载体pCambia1304-PbBP。
一种遗传工程化的宿主细胞,所述宿主细胞包括所述的梨转录因子PbBP的基因序列。
作为进一步地优选手段,所述的梨转录因子PbBP构建植物表达载体pCambia1304-PbBP质粒转化到感受态细胞中。
作为进一步地优选手段,所述宿主细胞采用农杆菌EHA105。
梨转录因子PbBP在调节植物木质素合成及细胞壁发育中的应用具体为:该基因对植物木质素合成及细胞壁发育起负调控作用,其超量表达可降低植株中木质素合成关键基因的表达量、木质素含量及细胞壁厚度。
本发明有益效果:梨转录因子PbBP具有负调控作用,可以直接通过抑制植物木质素合成基因的表达从而阻碍其木质素的生物合成,减缓细胞壁发育,降低木质化程度,以提高梨果肉品质,该基因为完善植物木质素调控网络提供新的证据,对水果品质改良(石细胞含量、果核大小及硬度等)以及作物其他关键农艺性状(如秸秆降解效率等)的改良均具有重要的理论及实践意义,同时也为利用基因工程调控梨石细胞含量提供了新的途径。
附图说明
图1为本发明的PbBP基因超量表达载体pCambia1304-PbBP构建图谱。
图2为本发明的PbBP基因超量表达拟南芥T3代植株组织化学染色结果图。
图3为本发明的PbBP基因超量表达拟南芥花序茎甲苯胺蓝染色结果图。
图4为本发明的PbBP基因超量表达拟南芥花序茎间苯三酚染色结果图。
图5为本发明的PbBP基因超量表达拟南芥花序茎木质素含量检测图。
图6为本发明的PbBP基因超量表达拟南芥中木质素合成关键基因的表达分析图。
图7为本发明的梨果实不同发育时期PbBP基因表达分析图。
图8为本发明的梨不同组织部位PbBP基因表达分析图。
具体实施方式
下面结合附图对本发明作进一步详细描述:
本发明的一种梨转录因子PbBP及其应用,下述实施例中所用方法如无特别说明均为常规方法,所用引物均由上海生工生物工程技术服务有限公司合成,测序由上海生工生物工程技术服务有限公司进行,pMD-18T、限制性内切酶Spe I和Bgl II、T4连接酶均购自宝日医生物技术(北京)有限公司(takara中国)。RNAprep Pure Plant Kit、FastQuant RTKit均购自TIANGEN公司。DNA胶回收试剂盒、质粒提取试剂盒购、X-Gluc均购自上海生工生物工程技术服务有限公司,方法均照说明书进行。梨为砀山酥梨。
一、PbBP基因克隆
选取砀山酥梨花后39天左右的果实,提取总RNA并反转录成cDNA。根据公布的梨基因组序列设计特异性扩增引物PbBP-F和PbBP-R,以cDNA为模板进行PCR扩增,PCR产物的回收,连接pMD-18T载体。
所述特异性扩增引物PbBP-F和PbBP-R为:
SEQ ID NO.3:PbBP-F:5’-ATGGAAGACTACAACAGTCAAATGGATCATG-3’
SEQ ID NO.4:PbBP-R:5’-TCATGGCCCGAGACGGTAGTGAACG-3’。
以反转录合成的cDNA为模板,利用引物PbBP-F和PbBP-R进行PCR扩增。反应体系如下:
PCR产物经1%琼脂糖凝胶电泳检测后,对目的片段进行回收,并连接到载体pMD-18T上,然后转化到大肠杆菌DH5α感受态细胞,阳性克隆鉴定后委托上海生工生物工程技术服务有限公司进行测序。
测序完成后利用DNAMAN软件进行比对,确保所获序列为目的序列。结果表明,所获基因大小为1194bp,其核苷酸序列及其编码的蛋白质的氨基酸序列如SEQ ID NO.1、SEQ IDNO.2所示。
二、PbBP超量表达载体和宿主细胞的构建
根据已经成功克隆的目的基因PbBP序列设计带有Bgl II和Spe I酶切位点的引物PbBP-ZH-F和PbBP-ZH-R,以已构建的pMD-18T-PbBP质粒为模板进行扩增,PCR产物经1%琼脂糖凝胶电泳检测后,对目的片段进行回收,并连接到载体pMD-18T上进行测序。
所述带有Bgl II和Spe I酶切位点的引物PbBP-ZH-F和PbBP-ZH-R为:
SEQ ID NO.5:PbBP-ZH-F:GAAGATCTGATGGAAGACTACAACAGTCAAATGGATCATG
SEQ ID NO.6:PbBP-ZH-R:GGACTAGTTGGCCCGAGACGGTAGTGAACG
如图1所示,用限制性内切酶Bgl II和Spe I分别对pMD-18T-PbBP质粒和植物表达载体pCambia1304质粒进行双酶切。酶切产物用1%的琼脂糖凝胶电泳,回收目的片段PbBP和植物表达载体pCambia1304。用T4连接酶将2个酶切后的目的片段连接并转化到大肠杆菌DH5α感受态细胞,重组质粒进行酶切验证后并进行测序验证,获得植物表达载体pCambia1304-PbBP。
将构建好的植物表达载体pCambia1304-PbBP通过电转化到农杆菌EHA105感受态细胞中,通过卡那霉素和利福平筛选出阳性菌株,获得宿主细胞。
三、拟南芥的遗传转化、筛选和鉴定
3.1制备侵染液:将所述宿主细胞活化后,用缓冲液重悬,获得侵染液,所述缓冲液为含有质量比为5%的蔗糖和0.02%的Silwet L-77的1/2MS液体培养基,所述侵染液的OD600值为0.8。
3.2将预转化的拟南芥苗的果荚和已开放的花朵全部剪掉,只留花蕾,将全部花蕾倒置浸在步骤3.1的侵染液中45s,再置于黑暗处培养一天,然后置于光照培养箱中继续培养,并控制光照时间为16h/d。
3.3每隔1周重复侵染一次,直至种子成熟,获得T0代种子。
3.4将T0代种子消毒后接种至含有50mg/L潮霉素的MS固体筛选培养基中,置于光照培养箱中培养,并控制光照时间为16h/d,非转基因植株黄化并停止生长。
待转基因幼苗长出2-3片真叶后,对其进行PCR检测,具体为:取少部分叶片,利用EasyPure Plant Genomic DNA Kit提取叶片的DNA,PCR鉴定时,利用目的基因的引物SEQID NO.3、SEQ ID NO.4进行检测。反应体系同PbBP基因克隆。在阳性转基因拟南芥中可扩增出大小1194bp的产物。
待转基因阳性株长出4-6片莲座叶后,移入营养土中继续培养并收获T1代种子。
3.5将T1代种子进行复筛,获得遗传稳定的T3代拟南芥阳性株。
3.6转基因拟南芥植株的GUS染色
取拟南芥阳性株T3代植株的幼苗,按照GUS染色试剂盒对其进行GUS染色,观察PbBP的组织表达情况,步骤如下:
(1)预处理:将拟南芥放于1.5mL离心管中,加入预冷的90%丙酮完全覆盖材料,常温处理20-30min。
(2)染色:用蒸馏水将材料漂洗干净后置于1.5mL离心管中,加入适量配置好的GUS染色工作液至完全覆盖材料,锡箔纸包好37℃过夜放置。
(3)洗脱:用95%乙醇进行洗脱,摇床轻摇。
(4)观察:肉眼或显微镜下观察,白色背景上的蓝色部分即为GUS表达位点。
结果发现:野生型拟南芥细胞中的各个部位都未有蓝色,而转化pCambia1304-PbBP的拟南芥幼苗的根、茎、叶都出现蓝色,表明PbBP在转基因拟南芥的各部位都表达(图2)。3.7拟南芥花序茎横切面染色和细胞壁厚度观察
分别取生长60d的超量表达T3代拟南芥与野生型拟南芥同位置的茎段,徒手切片,将切片分别进行甲苯胺蓝染色和Wiesner染色。Wiesner染色:切片用2%间苯三酚(溶于95%乙醇)染色5min,转入15%(v/v)HCl浸泡3min后直接封片观察。甲苯胺蓝染色:利用1%甲苯胺蓝浸泡后直接封片观察。甲苯胺蓝可以将植物细胞壁特异性染成蓝色,由图3可知,野生型拟南芥相比,PbBP超量表达植株花序茎中木质部和束间纤维细胞的细胞壁的厚度出现显著降低。Wiesner染色可以将木质素特异性染成紫红色,由图4可知,结PbBP超量表达植株的染色区域与野生型拟南芥相比减少且着色程度较浅,这说明PbBP超量表达植株的木质部和束间纤维区域的木质化程度降低。
3.8拟南芥花序茎木质素含量检测
利用紫外分光光度法(溴乙酰-冰乙酸),测定正常生长50d的野生型拟南芥(WT1~3)与超量表达PbBP拟南芥(PbBP-1~3)花序茎的木质素含量,测定均有三次生物学重复。显著性分析利用使用Statistical Program for Social Sciences软件(版本19.0,IBM,www.ibm.com)和Microsoft Excel 2010完成。测定结果显示,野生型拟南芥(WT1~3)中木质素含量分别为10.05%、10.07%、9.97%;超量表达PbBP拟南芥(PbBP1~3)中木质素含量分别为8.67%、8.73%、8.69%。可以发现超量表达PbBP拟南芥中木质素含量均显著低于野生型拟南芥(图5)。
3.9拟南芥木质素合成相关基因表达量检测
通过qRT-PCR对PbBP超量表达、野生型及bp-9突变体拟南芥中的木质素合成关键基因的表达量进行了分析(图6)。从图中可以看出,PbBP超量表达拟南芥株系中的木质素合成基因AtCAD4、AtCAD5、AtCCR和AtF5H的表达量显著降低,而与野生型和PbBP超量表达株系相比,基因敲除突变体株系bp-4中相应的木质素合成基因的表达量最高。表达模式分析结果表明PbBP通过抑制木质素合成关键基因的表达来阻碍木质素的生物合成及细胞壁的发育。
3.10 PbBP基因的时空表达模式分析
根据PbKNOX1即PbBP基因的核苷酸序列SEQ ID NO.1信息利用Primer Premier6.0软件设计其荧光定量引物:
SEQ ID NO.7:PbBP-Q-F:5’-TCATAAATCAAAGGAAGAGGCACTGGAA-3’
SEQ ID NO.8:PbBP-Q-R:5’-CAATGTCACGGAGTTGTTTAGGTCA-3’
表1荧光定量PCR反应体系
按照表1反应体系将各组分加入八连管中,以梨Tubulin(AccessionNo.AB239680.1)作为内参基因,每个样品进行三次生物学重复。以2-△△CT法计算各基因的相对表达水平。PCR反应程序为:98℃2min,98℃变性10s,60℃退火10s,68℃延伸30s,40个循环;后做65℃到99℃溶解曲线分析。所用仪器为GFX96Real-Time荧光定量PCR仪。
荧光定量的结果(图7、图8)显示,PbKNOX1(PbBP)的表达趋势总体上与木质素含量、石细胞含量呈负相关。在木质素和石细胞大量积累的时期(果实发育前期和中期)表达水平较低,在果实发育后期(102DAF)表达量升高。此外,PbBP在花、茎和芽中也有一定的转录水平。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
本发明不限于以上对实施例的描述,本领域技术人员根据本发明揭示的内容,在本发明基础上不必经过创造性劳动所进行的改进和修改,都应该在本发明的保护范围之内。
SEQUENCE LISTING
<110> 安徽农业大学
<120> 梨转录因子PbBP及其应用
<130> 2018/09/13
<160> 2
<170> PatentIn version 3.5
SEQ ID No.1
<210> 1
<211> 1194
<212> DNA
<213> Pabellonia incrassata
<400> 1
atggaagact acaacagtca aatggatcat gagagttcgg gtggtagggg aaacttcctc 60
tacgcctcac caaaccttgg aggaaattat gggagagctg caagtgatca ccagatgggg 120
atcaacacct ttcatcttca gtcaagcggc ggcggaggcg gcggcggcag cggtgatcag 180
tgtaattttc agtctccagg aacacaccca attaatgtga agaccgaagc caccacatca 240
cagcatggcc accaaaaatt tcagtacaac agcaataata ataatcatct tgtttcttca 300
tcaagagggc accaaccagt tgttcatcag ctacagaata atttgaatct tctaaacgac 360
gatcacagcc tgagctccaa cgaagttgaa gccatcaaag ccaagatcat cgcccaccct 420
cagtactcta acctcttgga agcttacatg gattgccaaa gggtgggagc tccgtctgat 480
gttatggctc ggctctcagt tgctaggcaa gagtttgagg cacgacagcg gtcttctggg 540
acttcaagag agacttcaaa agacccagaa ctggatcagt tcatggaagc ttactacgat 600
atgctggtta aatatcgtga agaactaaca aggccaatac aagaagccat ggatttcatg 660
aggaggattg aaactcagct taacatgctt ggaaataata ataatgctcc tccccttcgg 720
atcttctcac cctctgagga caagtgtgag ggaattggtt catctgaaga ggagcaggag 780
aatagtggtg gagaaacaga agtgcctgag attgatccaa gagctgaaga cagagagctc 840
aagaatcacc tgctgagaaa gtatagtggt tacttaagta gcctgaagca agagctttcc 900
aagaaaaaga agaaagggaa attgcccaaa gatgccaggc agaagctcct tagttggtgg 960
gagctacatt acaagtggcc atatccttcg gaatcggaga aggtggcttt ggcggagtct 1020
acgggtttgg atcagaaaca aataaacaat tggttcataa atcaaaggaa gaggcactgg 1080
aagccttccg aggacatgca gtttatggtg atggatggcc tacacccaca gaatgcagcc 1140
ctttatatgg atggacacta cataggtgac gttcactacc gtctcgggcc atga 1194
SEQ ID No.2
<210> 2
<211> 397
<212> PRT
<213> Pabellonia incrassata
<400> 2
Met Glu Asp Tyr Asn Ser Gln Met Asp His Glu Ser Ser Gly Gly Arg
1 5 10 15
Gly Asn Phe Leu Tyr Ala Ser Pro Asn Leu Gly Gly Asn Tyr Gly Arg
20 25 30
Ala Ala Ser Asp His Gln Met Gly Ile Asn Thr Phe His Leu Gln Ser
35 40 45
Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly Asp Gln Cys Asn Phe Gln
50 55 60
Ser Pro Gly Thr His Pro Ile Asn Val Lys Thr Glu Ala Thr Thr Ser
65 70 75 80
Gln His Gly His Gln Lys Phe Gln Tyr Asn Ser Asn Asn Asn Asn His
85 90 95
Leu Val Ser Ser Ser Arg Gly His Gln Pro Val Val His Gln Leu Gln
100 105 110
Asn Asn Leu Asn Leu Leu Asn Asp Asp His Ser Leu Ser Ser Asn Glu
115 120 125
Val Glu Ala Ile Lys Ala Lys Ile Ile Ala His Pro Gln Tyr Ser Asn
130 135 140
Leu Leu Glu Ala Tyr Met Asp Cys Gln Arg Val Gly Ala Pro Ser Asp
145 150 155 160
Val Met Ala Arg Leu Ser Val Ala Arg Gln Glu Phe Glu Ala Arg Gln
165 170 175
Arg Ser Ser Gly Thr Ser Arg Glu Thr Ser Lys Asp Pro Glu Leu Asp
180 185 190
Gln Phe Met Glu Ala Tyr Tyr Asp Met Leu Val Lys Tyr Arg Glu Glu
195 200 205
Leu Thr Arg Pro Ile Gln Glu Ala Met Asp Phe Met Arg Arg Ile Glu
210 215 220
Thr Gln Leu Asn Met Leu Gly Asn Asn Asn Asn Ala Pro Pro Leu Arg
225 230 235 240
Ile Phe Ser Pro Ser Glu Asp Lys Cys Glu Gly Ile Gly Ser Ser Glu
245 250 255
Glu Glu Gln Glu Asn Ser Gly Gly Glu Thr Glu Val Pro Glu Ile Asp
260 265 270
Pro Arg Ala Glu Asp Arg Glu Leu Lys Asn His Leu Leu Arg Lys Tyr
275 280 285
Ser Gly Tyr Leu Ser Ser Leu Lys Gln Glu Leu Ser Lys Lys Lys Lys
290 295 300
Lys Gly Lys Leu Pro Lys Asp Ala Arg Gln Lys Leu Leu Ser Trp Trp
305 310 315 320
Glu Leu His Tyr Lys Trp Pro Tyr Pro Ser Glu Ser Glu Lys Val Ala
325 330 335
Leu Ala Glu Ser Thr Gly Leu Asp Gln Lys Gln Ile Asn Asn Trp Phe
340 345 350
Ile Asn Gln Arg Lys Arg His Trp Lys Pro Ser Glu Asp Met Gln Phe
355 360 365
Met Val Met Asp Gly Leu His Pro Gln Asn Ala Ala Leu Tyr Met Asp
370 375 380
Gly His Tyr Ile Gly Asp Val His Tyr Arg Leu Gly Pro
385 390 395
Claims (1)
1.一种梨转录因子PbBP在调控植物木质素合成及细胞壁发育中的应用,其特征在于:所述梨转录因子PbBP的核苷酸序列如SEQ ID NO.1所示。
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Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1330719A (zh) * | 1998-12-14 | 2002-01-09 | 马克斯-普朗克-科学促进协会,柏林 | 影响植物的开花行为的手段和方法 |
CA2442755A1 (en) * | 2001-03-29 | 2002-10-10 | National Research Council Of Canada | Methods for modification of plant inflorescence architecture |
WO2002079463A2 (en) * | 2001-03-29 | 2002-10-10 | National Research Council Of Canada | Methods for modification of plant inflorescence architecture |
CN1869233A (zh) * | 2006-05-30 | 2006-11-29 | 清华大学 | 来源于棉花的基因启动子及其应用 |
US7663025B2 (en) * | 1999-03-23 | 2010-02-16 | Mendel Biotechnology, Inc. | Plant Transcriptional Regulators |
CN102492693A (zh) * | 2011-12-05 | 2012-06-13 | 华中师范大学 | 棉纤维特异性hb转录因子基因knat7的鉴定及应用 |
US8426678B2 (en) * | 2002-09-18 | 2013-04-23 | Mendel Biotechnology, Inc. | Polynucleotides and polypeptides in plants |
US8686226B2 (en) * | 1999-03-23 | 2014-04-01 | Mendel Biotechnology, Inc. | MYB-related transcriptional regulators that confer altered root hare, trichome morphology, and increased tolerance to abiotic stress in plants |
CN105255904A (zh) * | 2015-10-21 | 2016-01-20 | 中国科学院华南植物园 | 拟南芥转录因子基因bp在调控植物种子萌发中的应用 |
CN110331149A (zh) * | 2019-08-08 | 2019-10-15 | 浙江农林大学 | PagKNAT2/6b基因的应用和调控杨树株型的方法 |
CN110452915A (zh) * | 2019-09-16 | 2019-11-15 | 河南科技大学 | 葡萄VlKNOX基因促进细胞分裂素合成调控果实坐果的应用 |
CN111607599A (zh) * | 2020-07-03 | 2020-09-01 | 宁夏农林科学院农业生物技术研究中心(宁夏农业生物技术重点实验室) | 一种马铃薯KNOX转录因子StKNOX1基因、编码蛋白及其应用 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104946668A (zh) * | 2015-06-29 | 2015-09-30 | 湖南省植物保护研究所 | cry1Ia基因及应用、由其编码的Cry1Ia蛋白及制备方法和应用 |
CN105950632A (zh) * | 2016-05-25 | 2016-09-21 | 华中师范大学 | 棉花纤维发育相关的GhFSN1基因鉴定及应用 |
CN105936939B (zh) * | 2016-06-24 | 2019-12-13 | 东北林业大学 | 与长白落叶松木质素含量相关的snp标记及其应用 |
CN106754999A (zh) * | 2017-01-13 | 2017-05-31 | 安徽农业大学 | 一种棕色棉纤维原花青素转运相关的gst蛋白基因及其应用 |
CN108004262A (zh) * | 2018-01-15 | 2018-05-08 | 安徽农业大学 | 一种根癌农杆菌介导的平菇菌丝遗传转化方法 |
CN109207493B (zh) * | 2018-10-09 | 2022-04-12 | 安徽农业大学 | 一种梨漆酶基因PbLAC1及其载体、宿主细胞和应用 |
CN109156359B (zh) * | 2018-10-24 | 2022-04-12 | 安徽农业大学 | 一种砀山酥梨直接器官发生型再生途径的构建方法 |
CN110862444B (zh) * | 2019-12-18 | 2021-03-26 | 北京市农林科学院 | 一种玉米bm1基因突变体及该基因的分子标记和应用 |
CN112063631A (zh) * | 2020-09-17 | 2020-12-11 | 东北林业大学 | 毛果杨PtrLBD4-3基因及其编码蛋白和应用 |
-
2018
- 2018-09-29 CN CN201811148204.4A patent/CN109161553B/zh active Active
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1330719A (zh) * | 1998-12-14 | 2002-01-09 | 马克斯-普朗克-科学促进协会,柏林 | 影响植物的开花行为的手段和方法 |
US7663025B2 (en) * | 1999-03-23 | 2010-02-16 | Mendel Biotechnology, Inc. | Plant Transcriptional Regulators |
US8686226B2 (en) * | 1999-03-23 | 2014-04-01 | Mendel Biotechnology, Inc. | MYB-related transcriptional regulators that confer altered root hare, trichome morphology, and increased tolerance to abiotic stress in plants |
CA2442755A1 (en) * | 2001-03-29 | 2002-10-10 | National Research Council Of Canada | Methods for modification of plant inflorescence architecture |
WO2002079463A2 (en) * | 2001-03-29 | 2002-10-10 | National Research Council Of Canada | Methods for modification of plant inflorescence architecture |
US8426678B2 (en) * | 2002-09-18 | 2013-04-23 | Mendel Biotechnology, Inc. | Polynucleotides and polypeptides in plants |
CN1869233A (zh) * | 2006-05-30 | 2006-11-29 | 清华大学 | 来源于棉花的基因启动子及其应用 |
CN102492693A (zh) * | 2011-12-05 | 2012-06-13 | 华中师范大学 | 棉纤维特异性hb转录因子基因knat7的鉴定及应用 |
CN105255904A (zh) * | 2015-10-21 | 2016-01-20 | 中国科学院华南植物园 | 拟南芥转录因子基因bp在调控植物种子萌发中的应用 |
CN110331149A (zh) * | 2019-08-08 | 2019-10-15 | 浙江农林大学 | PagKNAT2/6b基因的应用和调控杨树株型的方法 |
CN110452915A (zh) * | 2019-09-16 | 2019-11-15 | 河南科技大学 | 葡萄VlKNOX基因促进细胞分裂素合成调控果实坐果的应用 |
CN111607599A (zh) * | 2020-07-03 | 2020-09-01 | 宁夏农林科学院农业生物技术研究中心(宁夏农业生物技术重点实验室) | 一种马铃薯KNOX转录因子StKNOX1基因、编码蛋白及其应用 |
Non-Patent Citations (7)
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