CN117051016A - 香蕉MaGWD1与MaPHO1复合体及其应用 - Google Patents
香蕉MaGWD1与MaPHO1复合体及其应用 Download PDFInfo
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Abstract
本发明提供了香蕉MaGWD1与MaPHO1复合体及其应用,其复合体由蛋白MaGWD1和蛋白MaPHO1相互作用形成。本发明还提供了与该复合体相关的基因组合、重组载体组合、宿主菌或表达盒以及单独基因和两基因组合的应用。本发明首次将MaGWD1基因和/或MaPHO1基因用于果肉淀粉降解及软化,研究发现MaGWD1基因、MaPHO1基因以及两基因组合同时作用均能够显著地促进果肉淀粉降解及软化,尤其是二者同时作用时,MaGWD1和MaPHO1能够互作形成蛋白复合体,促进果肉淀粉降解及软化效果最佳。本发明为香蕉生物育种提供了分子模块MaGWD1‑MaPHO1,为改良香蕉或其他植物软、甜、糯、营养风味品质提供了重要基因资源。
Description
技术领域
本发明属于基因工程技术领域,具体涉及香蕉MaGWD1与MaPHO1复合体及其应用。
背景技术
淀粉作为植物主要贮藏物质,主要以淀粉粒的形式贮藏于植物果实、种子、块根、块茎等贮藏器官。在叶片等营养器官中也有淀粉贮存,只是贮存于叶片中的淀粉属于暂时性淀粉,即白天合成,晚上降解,从而供给自身能量需求;而贮存于贮藏器官中的淀粉根据植物本身的特点其作用方式不同,例如香蕉、芒果等果实中的淀粉在采后成熟过程中,大多数淀粉转化成可溶性糖等风味物质;拟南芥种子中的淀粉在种子萌发时提供能量;而谷类作物种子中的淀粉几乎不降解 (Ritte et al., 2004; Skeffington et al., 2014;Wang et al., 2021; Uitdewilligen et al., 2022)。
香蕉属于典型的淀粉转化型果实 (张上隆和陈昆松, 2007; D′Hont et al.,2012; Wang et al., 2019; Miao et al., 2020)。刚采收时青香蕉果实中含有高达干重70%–80%的淀粉 (苗红霞等, 2013),是其产量性状和品质形成的物质基础;采收后随着果实成熟,淀粉快速降解与转化,至可食期淀粉含量仅为0.5%–3% (苗红霞等, 2013),形成软、香、甜、糯、营养等风味品质。但香蕉果实淀粉降解是涉及多种淀粉降解酶协同作用的复杂生物学过程。已报道α-淀粉酶 (α-amylase, AMY)、β-淀粉酶 (β-amylase, BAM)、异淀粉酶 (Isoamylase, ISA)、淀粉磷酸化酶 (Starch phosphorylase, PHO)等参与果实淀粉降解 (Junior et al., 2006; Jourda et al., 2016; Liu et al., 2021)。需要进一步开展相关研究。
发明内容
本发明的目的在于克服现有技术中的不足,提供一种香蕉MaGWD1与MaPHO1复合体及其应用。
本发明的第一个方面是提供一种蛋白复合体,所述蛋白复合体由蛋白MaGWD1和蛋白MaPHO1相互作用形成,所述蛋白MaPHO1的编码核苷酸序列如SEQ ID NO:1所示,所述蛋白MaGWD1的编码核苷酸序列如SEQ ID NO:2所示。
本发明的第二个方面是提供一种与本发明第一个方面所述的蛋白复合体相关的基因组合,包含编码所述蛋白MaGWD1的MaGWD1基因和编码蛋白MaPHO1的MaPHO1基因,所述MaPHO1基因的核苷酸序列如SEQ ID NO:1所示,所述MaGWD1基因的核苷酸序列如SEQ IDNO:2所示。
本发明的第三个方面是提供一种与本发明第一个方面所述的蛋白复合体相关的重组载体组合,所述重组载体组合包含编码所述蛋白MaGWD1的MaGWD1基因的重组载体和编码所述蛋白MaPHO1的MaPHO1基因的重组载体,所述MaPHO1基因的核苷酸序列如SEQ ID NO:1所示,所述MaGWD1基因的核苷酸序列如SEQ ID NO:2所示。
其中,所述重组载体原始载体可以采用基因重组领域中常用的载体,例如病毒、质粒等。本发明对此不进行限定。在本发明的一个具体实施方式中,所述原始载体采pGBKT7载体、pGADT7载体、pCAMBIA1304载体,但应当理解的是,本发明还可以采用其他质粒、或者病毒等。
本发明的第四个方面是提供含有本发明第二个方面所述基因组合的宿主菌或表达盒。
本发明的第五个方面是提供如本发明第一个方面所述的蛋白复合体、或者如本发明第二个方面所述的基因组合、或者如本发明第三个方面所述的重组载体组合、或者本发明第四个方面所述宿主菌或表达盒在促进果肉淀粉降解、和/或果肉软化中的应用。
在本发明一个具体的实施方式中,所述果肉为香蕉的果肉。应当理解的是,此为举例说明,本发明第一个方面所述的蛋白复合体、或者如本发明第二个方面所述的基因组合、或者如本发明第三个方面所述的重组载体组合、或者本发明第四个方面所述宿主菌或表达盒也可以应用于促进其他植物的果肉淀粉降解、和/或果肉软化。
本发明的第六个方面是提供MaGWD1基因、或者MaGWD1基因编码的蛋白MaGWD1、或者含有所述MaGWD1基因的重组载体或宿主菌或表达盒在促进果肉淀粉降解、和/或果肉软化中的应用,所述MaGWD1基因的核苷酸序列如SEQ ID NO:2所示。
优选地,MaGWD1与MaPHO1互作来促进果肉淀粉降解、和/或果肉软化,MaPHO1基因的核苷酸序列如SEQ ID NO:1所示。
在本发明一个具体的实施方式中,所述果肉为香蕉的果肉。应当理解的是,此为举例说明,MaGWD1基因、或者MaGWD1基因编码的蛋白MaGWD1、或者含有所述MaGWD1基因的重组载体或宿主菌或表达盒也可以应用于促进其他植物的果肉淀粉降解、和/或果肉软化。
本发明的第七个方面是提供MaPHO1基因、或者MaPHO1基因编码的蛋白MaPHO1、或者含有所述MaPHO1基因的重组载体或宿主菌或表达盒在促进果肉淀粉降解、和/或果肉软化中的应用,所述MaPHO1基因的核苷酸序列如SEQ ID NO:1所示。
优选地,MaGWD1与MaPHO1互作来促进果肉淀粉降解、和/或果肉软化,MaGWD1基因的核苷酸序列如SEQ ID NO:2所示。
在本发明一个具体的实施方式中,所述果肉为香蕉的果肉。应当理解的是,此为举例说明,MaPHO1基因、或者MaPHO1基因编码的蛋白MaPHO1、或者含有所述MaPHO1基因的重组载体或宿主菌或表达盒也可以应用于促进其他植物的果肉淀粉降解、和/或果肉软化。
本发明的第八个方面是提供一种促进果肉淀粉降解、和/或果肉软化的方法,采用MaGWD1基因和/或MaPHO1基因转化果实,所述MaPHO1基因的核苷酸序列如SEQ ID NO:1所示,所述MaGWD1基因的核苷酸序列如SEQ ID NO:2所示。
在本发明一个具体的实施方式中,所述果肉为香蕉的果肉。应当理解的是,此为举例说明,本方法也可以用于促进其他植物的果肉淀粉降解、和/或果肉软化。
本发明首次将MaGWD1基因和/或MaPHO1基因用于果肉淀粉降解及软化,研究发现MaGWD1基因、MaPHO1基因以及两基因组合同时作用均能够显著地促进果肉淀粉降解及软化,尤其是二者同时作用时,MaGWD1和MaPHO1能够互作形成蛋白复合体,促进果肉淀粉降解及软化效果最佳。本发明为香蕉生物育种提供了分子模块MaGWD1-MaPHO1,为改良香蕉或其他植物软、甜、糯、营养风味品质提供了重要基因资源。
附图说明
图1为MaPHO1和MaGWD1酵母双杂交结果图,A:MaPHO1和MaGWD1两个蛋白在二缺培养基上正常生长;B:MaPHO1和MaGWD1两个蛋白在四缺培养基上长出蓝色斑点;pGBKT7-pGADT7:阳性对照;pGBKT7-53+pGADT7-T-antigen:阴性对照;pGBKT7-MaPHO1+pGADT7-MaGWD1:实验组; SD-Leu-Trp:二缺培养基;SD-Ade-His-Leu-Trp+x-α-gal:四缺培养基并添加了x-α-gal ;10-1、10-2、10-3、10-4:菌液梯度稀释倍数。
图2为MaPHO1和MaGWD1蛋白GST pull-down验证,GST:GST 蛋白;MaPHO1-GST:带GST标签的MaPHO1蛋白;MaGWD1-His:带His标签的MaGWD1蛋白;Anti-His-GST:以His和GST抗体进行western blot分析;Anti-His:以His抗体进行western blot分析;Anti-GST:以GST抗体进行western blot分析;Input:加入;Pull-down:蛋白挂柱下拉。
图3为瞬时过表达MaPHO1和MaGWD1促进了香蕉果实软化及淀粉降解,A:单独注射空载体 (对照)和MaPHO1后香蕉果实表型;B:单独注射MaPHO1和共同注射MaPHO1和MaGWD1后香蕉果实表型;C:香蕉果实硬度变化;D:总淀粉含量变化; pCAMBIA3300:空载体;pCAMBIA3300-MaPHO1:瞬时过表达MaPHO1基因;pCAMBIA3300-MaGWD1:瞬时过表达MaGWD1基因;pCAMBIA3300-MaPHO1+ pCAMBIA3300-MaGWD1:瞬时共表达MaPHO1和MaGWD1基因; *(p <0.05) 达到显著差异水平,**(p < 0.01) 达到极显著差异水平。
具体实施方式
下面参照附图,结合具体的实施例对本发明作进一步的说明,以更好地理解本发明。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:基因的克隆
以巴西蕉果实cDNA为模板,以5’-ATGTCGTCGTCCCCGTTCCCGTCTC-3’和5’-TCATGGCAAGACGACAGGCTTTATG -3’ 为引物, 通过PCR方法扩增获得的一种含有碱基序列为2,997 bp,其序列如SEQ ID No.1所示。
PCR反应体系如下:
PCR扩增程序如下:
2、香蕉MaGWD1基因的克隆
以巴西蕉果实cDNA为模板,以5’-ATGAGCAATACTGTTGGACACACC T-3’ 和5’-TCACATTTGCGGTCTTGTTTGGAC-3’ 为引物, 通过PCR方法扩增获得的一种含有碱基序列为4,437 bp,其序列如SEQ ID No.2所示。
PCR反应体系如下:
PCR扩增程序如下:
实施例3:重组载体的构建
以巴西蕉果实cDNA为模板,以引物GCTGCAGCATGTCGTCGTCCCCG TTCCCGTCTC(划线为Pst I酶切位点)和TTGCGGCCGCAATCATGGCAA GACGACAGGCTTTATG(划线为Not I酶切位点),进行PCR扩增。反应体系为2×Taq Mix 10 µL,Primer-F 0.5 µL,Primer-R 0.5 µL,cDNA模板,0.5 µL ddH2O 8.5 µL。反应程序为94℃ 4 min;94℃ 40 s,57℃ 2.5 min,72℃80 s,共35 cycles;72℃ 10 min。扩增产物经Pst I和Not I双酶切后连接至经相同酶切后的pGBKT7表达载体,得到pGBKT7-MaPHO1载体。
以巴西蕉果实cDNA为模板,以引物CGAGCTCGATGAGCAATACTGTT GGACACACCT(划线为Sac I酶切位点)和CCTCGAGGTCACATTTGCGGT CTTGTTTGGAC(划线为Xho I酶切位点),进行PCR扩增。反应体系为2×Taq Mix 10 µL,Primer-F 0.5 µL,Primer-R 0.5 µL,cDNA模板0.5 µL,ddH2O 8.5 µL。反应程序为94℃ 4 min;94℃ 40 s,58℃ 3 min,72℃ 80 s,共35cycles;72℃ 10 min。扩增产物经Sac I和Xho I双酶切后连接至经相同酶切后的pGBKT7表达载体,得到pGADT7-MaGWD1载体。
以巴西蕉果实cDNA为模板,以引物GGGGTACCCCATGTCGTCGTCCCCGTTCCCG TCTC(划线为Kpn I酶切位点)和CGAGCTCGTCATGGCAAGACGACAGGCTTTATG(划线为Sac I酶切位点),进行PCR扩增。反应体系为2×Taq Mix 10 µL,Primer-F 0.5 µL,Primer-R 0.5 µL,cDNA模板,0.5 µL ddH2O 8.5 µL。反应程序为94℃ 4 min;94℃ 40 s,57℃ 2.5 min,72℃ 80 s,共35 cycles;72℃ 10 min。扩增产物经Kpn I和Sac I双酶切后连接至经相同酶切后的pCAMBIA3300表达载体,得到pCAMBIA3300-MaPHO1载体。
以巴西蕉果实cDNA为模板,以引物 CCCCCGGGGGATGAGCAATACTGTTGGACAC ACCT(划线为Sma I酶切位点)和GGGGTACCCC TCACATTTGCGGTCTTGTTTGGAC(划线为Kpn I酶切位点),进行PCR扩增。反应体系为2×Taq Mix 10 µL,Primer-F 0.5 µL,Primer-R 0.5 µL,cDNA模板 0.5 µL,ddH2O 8.5 µL。反应程序为94℃ 4 min;94℃ 40 s,58℃ 3 min,72℃80 s,共35 cycles;72℃ 10 min。扩增产物经Sma I和Kpn I双酶切后连接至经相同酶切后的pCAMBIA3300表达载体,得到pCAMBIA3300-MaGWD1载体。
实施例4:酵母双杂交实验
阳性对照:pGBKT7载体与pGADT7载体共转化酵母菌株AH109;
阴性对照:pGBKT7-53载体与pGADT7-T-antigen载体(Clontech公司购买)共转化酵母菌株AH109。
实验组:pGBKT7-MaPHO1载体和pGADT7-MaGWD1载体共转化酵母菌株AH109。
阳性对照、阴性对照和实验组分别共转化酵母菌株AH109,30℃培养3 d。挑取单菌落,加入10 mL 0.5×YPDA/Kan+ 液体培养基,200 rpm,30℃培养3 d。将取出的100 μL菌液按1: 10、1: 100、1:1000、1:10000比例稀释后,先后接种在二缺培养基 (SD/-Leu/-Trp)和四缺培养基 (SD/-Ade/-His/-Leu/-Trp/Aba/x-α-gal) 上;通过蓝色斑点筛选,鉴定MaPHO1和MaGWD1之间是否互作。
结果如图1所示,在二缺培养基上,阳性对照 (pGBKT7-pGADT7)、阴性对照(pGBKT7-53+pGADT7-T-antigen)和实验组(pGBKT7-MaPHO1+pGADT7-MaGWD1) 均能正常生长,说明转进去的两两质粒上均具有合成亮氨酸(Leu)和色氨酸(Trp)两种氨基酸的基因,所以在二缺培养基上能够正常生长。在四缺培养基上,阳性对照 (pGBKT7-pGADT7) 和实验组(pGBKT7-MaPHO1+pGADT7-MaGWD1) 均能长出蓝色斑点,说明MaPHO1和MaGWD1两个蛋白能够互作,组合成一个完整的复合体后,这个复合体会催化x-α-gal发生转化,所以在四缺培养基上生长出蓝色斑点;阴性对照(pGBKT7-53+pGADT7-T-antigen) 没有长出蓝色斑点,说明两两蛋白不能互作,就不能生长。
实施例4:GST pull-down实验
(1)GST-MaPHO1融合蛋白的诱导表达与纯化
以巴西蕉果实cDNA为模板,以引物CCTCGAGGATGTCGTCGTCCCCGTTCCCGTCTC(划线为Xho I酶切位点)和TTGCGGCCGCAATCATGGCAA GACGACAGGCTTTATG(划线为Not I酶切位点),进行PCR扩增。反应体系为2×Taq Mix 10 µL,Primer-F 0.5 µL,Primer-R 0.5 µL,cDNA模板,0.5 µL ddH2O 8.5 µL。反应程序为94℃ 4 min;94℃ 40 s,57℃ 2.5 min,72℃ 80 s,共35 cycles;72℃ 10 min。扩增产物经Xho I和Not I双酶切后连接至经相同酶切后的pGEX-4T-3表达载体,得到pGEX-4T-3-MaPHO1载体。
将构建好的pGEX-4T-3-MaPHO1质粒转化大肠杆菌Rosetta宿主菌。从转化了pGEX-4T-3-MaPHO1的阳性菌落中挑取单菌落,接种于5 mL含100 mg/L Amp和34 mg/L Chl的LB液体培养基中,37℃摇菌培养过夜。应用终浓度为0.2 mmol/L的IPTG,28℃诱导表达过夜。富集诱导表达的大肠杆菌,超声后上清用Glutathione-Sepharose 4B 亲和层析柱纯化获得大量诱导表达的GST-MaPHO1融合蛋白,用于下一步GST pull-down实验。
(2)MaGWD1-His融合蛋白的诱导表达与纯化
以巴西蕉果实cDNA为模板,以引物 TTGCGGCCGCAAATGAGCAATACTGTTGGAC ACACCT(划线为Not I酶切位点)和CCCATATGGGTCACAT TTGCGGTCTTGTTTGGAC(划线为Nde I酶切位点),进行PCR扩增。反应体系为2×Taq Mix 10 µL,Primer-F 0.5 µL,Primer-R 0.5 µL,cDNA模板 0.5 µL,ddH2O 8.5 µL。反应程序为94℃ 4 min;94℃ 40 s,58℃ 3 min,72℃80 s,共35 cycles;72℃ 10 min。扩增产物经Not I和Nde I双酶切后连接至经相同酶切后的pET28表达载体,得到pET28-MaGWD1载体。
将构建好的pET28-MaGWD1质粒转化BL21宿主菌,用含有50 mg/L Kan的LB液体培养基培养转化有pET28-MaGWD1的阳性单克隆菌落,应用终浓度为0.5 mmol/L 的ITPG,28℃诱导表达过夜。富集诱导表达的大肠杆菌,超声后上清用组氨酸标记亲和层析柱纯化获得大量诱导表达的MaGWD1-His融合蛋白,用于下一步GST pull-down实验。
(3)GST-MaPHO1蛋白和MaGWD1-His蛋白的GST pull-down实验
蛋白GST- MaPHO1和蛋白MaGWD1-His进行GST Pull-Down实验,来验证蛋白GST-MaPHO1与蛋白MaGWD1-His是否具有相互作用。GST蛋白作为阴性对照。将Input组各样品与GST Magarose Beads反应后,用洗脱液洗脱后,进行WB鉴定,从而判断验证蛋白GST-MaPHO1与蛋白MaGWD1-His是否能够形成复合体,以及两两蛋白是否存在相关作用。
实验具体分组如下:
表1 GST-MaPHO1和MaGWD1-His实验具体分组
编号 | 1 | 2 | 3 | 4 |
GST蛋白 | - | - | + | - |
GST-MaPHO1 | + | - | - | + |
MaGWD1-His | - | + | + | + |
① 平衡磁珠(GST Magarose Beads)
混匀GST Magarose Beads,彻底重悬磁珠;各取100 µL 50% 磁珠悬浮液到4个离心管中,2000 r/min离心2 min弃上清;用500 µL PBS重悬磁珠,2000 r/min离心2 min移除上清,重复3次。
② 蛋白互作
按照表1加入相应的样品,并进行编号。混合后将平衡后的GST Magarose Beads分别加入到Input混合的样品中,在4℃旋转混匀孵育4 h。2000 r/min离心5 min,吸取上清。用500 µL PBS洗涤磁珠,2000 r/min离心2 min,弃上清,重复3次。
③ GST Magarose Beads洗脱
每管加入100 µL洗脱液。4℃旋转混匀孵育30 min。2000 r/min离心2 min取上清,置于冰上。取20 µL样品进行WB检测。
④ Western Blot
取自备好的样品15% SDS-PAGE胶上样。上样完毕后,聚丙烯酰胺凝胶先100 V跑完积层胶,再将电压升至130 V直到电泳结束。电泳结束后,取下凝胶进行转膜,恒流300 mA转膜,约为1 h。电转结束后,取下膜后先用PBST洗涤4次,每次5 min。将膜置于5%脱脂奶粉封闭液中封闭37℃ 1 h。用PBST稀释一抗,膜在一抗稀释液中4℃过夜。次日将膜取出后用PBST 洗膜4次,每次5 min。用含5%脱脂奶粉的封闭液稀释二抗。膜在二抗中37℃反应1 h。反应完毕后,把膜取出后置于干净的盒子中洗膜4次,每次5 min。ECL显影,曝光。
结果如图2所示,与GST标签融合表达的MaPHO1蛋白,而不是单独的GST蛋白,能够与带His标签的MaGWD1蛋白挂柱,洗脱获得两者互作的蛋白复合体;以GST和His抗体做western blot检测,能够看到明显的条带,说明MaPHO1和MaGWD1存在相互作用。
实施例5:转化香蕉实验
(1)pCAMBIA3300-MaPHO1载体的农杆菌转化
取200 µL冰浴上融化的根癌农杆菌GV3101感受态细胞,加入pCAMBIA3300-MaPHO1重组质粒2 µg,轻轻混匀,在冰浴中放置30 min;转入液氮中冷冻3 min,迅速置37 ℃水浴中温育5 min;加入800 µL YEP液体培养基,28 ℃,250 rpm预培养4-5 h;吸取300 µL菌液至含有50 mg/L Kan 的YEP固体选择培养基上,均匀涂布于整个平板;将平板置于28 ℃至液体被吸收,倒置平板,28 ℃培养2-3 d,挑选单菌落,验证检测,将转化正确的农杆菌菌液用于下一步实验。
(2)pCAMBIA3300-MaGWD1载体的农杆菌转化
取200 µL冰浴上融化的根癌农杆菌GV3101感受态细胞,加入pCAMBIA3300-MaGWD1重组质粒2 µg,轻轻混匀,在冰浴中放置30 min;转入液氮中冷冻3 min,迅速置37 ℃水浴中温育5 min;加入800 µL YEP液体培养基,28 ℃,250 rpm预培养4-5 h;吸取300 µL菌液至含有50 mg/L Kan 的YEP固体选择培养基上,均匀涂布于整个平板;将平板置于28 ℃至液体被吸收,倒置平板,28 ℃培养2-3 d,挑选单菌落,验证检测,将转化正确的农杆菌菌液用于下一步实验。
(3)根癌农杆菌介导香蕉果实遗传转化
在超净工作台,取采收期的香蕉果实6根, 20%次氯酸钠溶液浸泡15 min,浸泡期间充分晃动,用无菌水冲洗3次。将已经转化pCAMBIA3300-MaPHO1重组载体与pCAMBIA3300-MaGWD1的根癌农杆菌GV3101菌液按照1:1的体积比加入到10 mL含有50 mg/L Kan的 YEP液体培养基中过夜活化培养;吸取活化培养的菌液1 mL到新的50 mL含有50 mg/L Kan的YEP液体培养基中培养至OD600 = 0.6;将所需浓度的菌液于超净工作台上转移到50 mL无菌离心管中,4 ℃,6000 rpm离心5 min,弃去上清液,加入等体积 (离心前菌液体积) 的MS液体培养基将菌体重新悬浮;将菌液转移到100 mL无菌三角瓶中,加入0.1%体积 (重悬菌液体积) 的乙酰丁香酮 (AS),充分混匀。取4 mL重悬好的菌液,然后用注射器将农杆菌菌液注射至香蕉果实,25 ℃培养3 d;将共培养的香蕉果实用于淀粉含量及果实硬度的检测。
(4)淀粉含量的测定方法
取注射部位的香蕉果肉浸入0.5%亚硫酸氢钠溶液中护色10 min,于40 ℃干燥20-24 h,磨粉。取100 mg香蕉粉于15 mL离心管中, 加入5 mL的80 %乙醇,充分振荡混匀。4000rpm离心5 min后弃上清,沉淀用5 mL蒸馏水悬浮洗涤一次, 再次离心后沉淀用5 mL的80 %Ca(NO3)2悬浮,沸水浴中放置10 min,4000 rpm离心5 min后将上清液转入20 mL容量瓶中。沉淀用80 %Ca(NO3)2重新提取2次,合并提取液,用80 %Ca(NO3)2定容至20 mL。取淀粉液0.5mL,用80 %Ca(NO3)2补充至2 mL,加入100 μL的0.01 N I2-KI 溶液,混匀后于620 nm波长下测定吸光度值,代人标准曲线即可计算出样品淀粉含量。
标准曲线的制作方法:取七个容量瓶分别加入0、100、200、500、1000、1500、2000 μL的100 μg/mL淀粉标准溶液,用80 %Ca(NO3)2补充至2 mL,加入100 μL的I2-KI溶液,混匀后于620 nm波长下测定吸光度值,以淀粉溶液浓度为纵坐标,吸光度值为横坐标,绘制标准曲线。
淀粉含量(mg/g)=(X×2×Vt)/(Vu×W×1000),其中X—标准曲线上查得的样品淀粉浓度(μg/mL),2—每支测定试管中样品总体积(mL),Vt—样品总体积(mL),Vu—每支测定试管中加入样品原液的体积(mL),W—样品质量(g)。
(5)香蕉果实硬度的测定方法
采用GY-3型(杭州托普仪器有限公司)水果硬度计(直径5 mm,长度10 mm)测定果实硬度,将香蕉果实注射孔附近的1 cm2果皮削去,硬度计垂直于被测果肉表面,压头均匀压入果肉,当压头压到刻度线10 mm处停止,指针指示的读数即为果实的硬度,取3次平均值,硬度用kg/cm2表示。
结果如图3所示,瞬时过表达pCAMBIA3300-MaPHO1和pCAMBIA3300-MaGWD1组合,果实呈现成熟速度加快的表型,果肉明显变软(图3B);且pCAMBIA3300-MaPHO1和pCAMBIA3300-MaGWD1组合成熟速度及果肉变软程度强于单独瞬时过表达pCAMBIA3300-MaPHO1 或pCAMBIA3300-MaGWD1(图3A)。
与对照空载体pCAMBIA3300相比较,瞬时过表达pCAMBIA3300-MaPHO1、pCAMBIA3300-MaGWD1果肉硬度从0.83 kg/cm2下降至0.51 kg/cm2、0.56 kg/cm2,分别下降了1.63倍、1.48倍,达到显著差异水平;与对照空载体pCAMBIA3300相比较,瞬时过表达pCAMBIA3300-MaGWD1和pCAMBIA3300-MaPHO1组合果肉硬度从0.83 kg/cm2下降至0.14 kg/cm2,下降了5.93倍,达到极显著差异水平(图3C)。
与对照空载体pCAMBIA3300,瞬时过表达pCAMBIA3300-MaPHO1、瞬时过表达pCAMBIA3300-MaGWD1淀粉含量从211.79 mg/g下降至122.65 mg/g、123.92 mg/g,分别下降了1.73倍、1.71倍,达到显著差异水平(图3D);与对照空载体pCAMBIA3300,瞬时过表达pCAMBIA3300-MaPHO1和pCAMBIA3300-MaGWD1组合淀粉含量从211.79 mg/g下降至28.87mg/g,下降了7.34倍,达到极显著差异水平(图3D)。
以上对本发明的具体实施例进行了详细描述,但其只作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对该实用进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (6)
1.一种蛋白复合体,其特征在于,所述蛋白复合体由蛋白MaGWD1和蛋白MaPHO1相互作用形成,所述蛋白MaPHO1的编码核苷酸序列如SEQ ID NO:1所示,所述蛋白MaGWD1的编码核苷酸序列如SEQ ID NO:2所示。
2.一种与权利要求1所述的蛋白复合体相关的基因组合,其特征在于,包含编码所述蛋白MaGWD1的MaGWD1基因和编码蛋白MaPHO1的MaPHO1基因,所述MaPHO1基因的核苷酸序列如SEQ ID NO:1所示,所述MaGWD1基因的核苷酸序列如SEQ ID NO:2所示。
3.一种与权利要求1所述的蛋白复合体相关的重组载体组合,其特征在于,所述重组载体组合包含编码所述蛋白MaGWD1的MaGWD1基因的重组载体和编码所述蛋白MaPHO1的MaPHO1基因的重组载体,所述MaPHO1基因的核苷酸序列如SEQ ID NO:1所示,所述MaGWD1基因的核苷酸序列如SEQ ID NO:2所示。
4.含有权利要求2所述基因组合的宿主菌或表达盒。
5.如权利要求1所述的蛋白复合体、或者如权利要求2所述的基因组合、或者如权利要求3所述的重组载体组合、或者权利要求4所述宿主菌或表达盒在促进香蕉果肉淀粉降解、和/或香蕉果肉软化中的应用。
6.MaGWD1基因、或者MaGWD1基因编码的蛋白MaGWD1、或者含有所述MaGWD1基因的重组载体或宿主菌或表达盒、 或者MaPHO1基因、或者MaPHO1基因编码的蛋白MaPHO1、或者含有所述MaPHO1基因的重组载体或宿主菌或表达盒在促进香蕉果肉淀粉降解、和/或香蕉果肉软化中的应用,其特征在于,MaGWD1与MaPHO1互作来促进果肉淀粉降解、和/或果肉软化。
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