生物技术通报 ›› 2020, Vol. 36 ›› Issue (11): 21-29.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0510
郝小花1,2(), 戴佳利2, 暨文劲2, 黄丹2, 李东屏2, 田连福2()
收稿日期:
2020-04-30
出版日期:
2020-11-26
发布日期:
2020-11-20
作者简介:
郝小花,女,博士,研究方向:植物生态学;E-mail: 基金资助:
HAO Xiao-hua1,2(), DAI Jia-li2, JI Wen-jin2, HUANG Dan2, LI Dong-ping2, TIAN Lian-fu2()
Received:
2020-04-30
Published:
2020-11-26
Online:
2020-11-20
摘要:
水稻低镉基因(Low cadmium,LCD)参与水稻籽粒镉(Cd)的积累,筛选与LCD互作的蛋白有利于揭示LCD介导的籽粒Cd积累调控的分子机制。以粳稻日本晴为试验材料构建酵母双杂交膜系统和核系统cDNA文库,以LCD为诱饵蛋白,分别进行膜系统和核系统筛选,获得29个与LCD互作的候选蛋白,并对这些基因进行gene ontology(GO)注释分析。进一步通过一对一互作验证,获得21个互作蛋白,包括膜系统蛋白16个,核系统蛋白5个,其中,6个蛋白与物质转运相关,5个蛋白与植物耐逆相关,4个蛋白与植物发育响应相关,6个蛋白与细胞生理代谢相关。
郝小花, 戴佳利, 暨文劲, 黄丹, 李东屏, 田连福. 水稻籽粒低镉蛋白LCD互作蛋白的筛选与鉴定[J]. 生物技术通报, 2020, 36(11): 21-29.
HAO Xiao-hua, DAI Jia-li, JI Wen-jin, HUANG Dan, LI Dong-ping, TIAN Lian-fu. Screening and Identification of LCD-interacting Proteins in Rice[J]. Biotechnology Bulletin, 2020, 36(11): 21-29.
引物名称 | 引物序列(5'-3') | 酶切位点 |
---|---|---|
pBT3-N-F | GGCCATTACGGCCATGGTGGAAACCACAGTT | Sfi I |
pBT3-N-R | GGCCGAGGCGGCCTTTTTTACAAGGCCACTT | Sfi I |
pGBKT7-F | GGAATTCATGGTGGAAACCACAGTTGATGTT | EcoR I |
pGBKT7-R | CGGGATCCGTTTTTTACAAGGCCACTTCCAG | BamH I |
表1 构建诱饵载体的引物序列
引物名称 | 引物序列(5'-3') | 酶切位点 |
---|---|---|
pBT3-N-F | GGCCATTACGGCCATGGTGGAAACCACAGTT | Sfi I |
pBT3-N-R | GGCCGAGGCGGCCTTTTTTACAAGGCCACTT | Sfi I |
pGBKT7-F | GGAATTCATGGTGGAAACCACAGTTGATGTT | EcoR I |
pGBKT7-R | CGGGATCCGTTTTTTACAAGGCCACTTCCAG | BamH I |
引物名称 | 引物序列(5'-3') |
---|---|
pPR3-N-F | GTCGAAAATTCAAGACAAG |
pPR3-N-R | AAGCGTGACATAACTAATTA |
pGADT7-F | GGAGTACCCATACGACGTACC |
pGADT7-R | TATCTACGATTCATCTGCAGC |
表2 扩增文库载体插入片段的引物序列
引物名称 | 引物序列(5'-3') |
---|---|
pPR3-N-F | GTCGAAAATTCAAGACAAG |
pPR3-N-R | AAGCGTGACATAACTAATTA |
pGADT7-F | GGAGTACCCATACGACGTACC |
pGADT7-R | TATCTACGATTCATCTGCAGC |
组合 | 质粒1 | 质粒2 | 目的 | 缺陷培养基 | 生长状态 |
---|---|---|---|---|---|
1 | pBT3-N-LCD | - | 自激活检测 | SD-Leu SD-Lue-Trp | √ × |
2 | pBT3-N-LCD | pOst-Nub1 | 阳性对照 | SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade | √ √ √ |
3 | pBT3-N-LCD | pPR3-N | 阴性对照 | SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade | √ ×/少量 × |
表3 DUALmembrane系统诱饵蛋白LCD自激活体系
组合 | 质粒1 | 质粒2 | 目的 | 缺陷培养基 | 生长状态 |
---|---|---|---|---|---|
1 | pBT3-N-LCD | - | 自激活检测 | SD-Leu SD-Lue-Trp | √ × |
2 | pBT3-N-LCD | pOst-Nub1 | 阳性对照 | SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade | √ √ √ |
3 | pBT3-N-LCD | pPR3-N | 阴性对照 | SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade | √ ×/少量 × |
序号 | 基因ID | 功能预测 |
---|---|---|
1 | LOC_Os01g67250 | Rad21 / Rec8 like protein |
2 | LOC_Os10g34614 | csAtPR5 |
3 | LOC_Os07g39900 | interferon-related developmental regulator(IFRD) |
4 | LOC_Os08g01780 | OsIAA25-Auxin-responsive,Aux/IAA gene family member |
5 | LOC_Os02g35560 | OsFBX53 - F-box domain containing protein |
6 | LOC_Os06g45120 | ATP synthase |
7 | LOC_Os03g38020 | MOB激酶家族,mps one binder kinase activator-like 1A |
8 | LOC_Os03g17140 | expressed protein |
9 | LOC_Os08g42050 | emp24/gp25L/p24 family protein,transporter activity |
10 | LOC_Os12g32380 | 40S ribosomal protein S29 |
11 | LOC_Os05g03780 | 金属耐受蛋白基因(OsMTP1) |
12 | LOC_Os07g44180 | 低温诱导的小分子量完整膜蛋白基因(OsLti6a) |
13 | LOC_Os09g30418 | heat shock protein,HSP |
14 | LOC_Os01g69220 | exostosin family domain containing protein |
15 | LOC_Os01g38970 | carbamoyl-pHospHate synthase large chain |
16 | LOC_Os03g57790 | 泛素结合酶,UBC |
17 | LOC_Os08g41990 | Aminotransferase |
18 | LOC_Os10g08550 | 2-磷酸甘油酸水解酶,enolase |
19 | LOC_Os06g08170 | MFS transporter |
20 | LOC_Os11g25980 | phospholipid-transporting ATPase2 |
21 | LOC_Os08g44280 | L-异天冬氨酸甲基转移酶(OsPIMT1) |
22 | LOC_Os02g33500 | threonyl-tRNA synthetase,putative |
表4 候选蛋白的功能分析
序号 | 基因ID | 功能预测 |
---|---|---|
1 | LOC_Os01g67250 | Rad21 / Rec8 like protein |
2 | LOC_Os10g34614 | csAtPR5 |
3 | LOC_Os07g39900 | interferon-related developmental regulator(IFRD) |
4 | LOC_Os08g01780 | OsIAA25-Auxin-responsive,Aux/IAA gene family member |
5 | LOC_Os02g35560 | OsFBX53 - F-box domain containing protein |
6 | LOC_Os06g45120 | ATP synthase |
7 | LOC_Os03g38020 | MOB激酶家族,mps one binder kinase activator-like 1A |
8 | LOC_Os03g17140 | expressed protein |
9 | LOC_Os08g42050 | emp24/gp25L/p24 family protein,transporter activity |
10 | LOC_Os12g32380 | 40S ribosomal protein S29 |
11 | LOC_Os05g03780 | 金属耐受蛋白基因(OsMTP1) |
12 | LOC_Os07g44180 | 低温诱导的小分子量完整膜蛋白基因(OsLti6a) |
13 | LOC_Os09g30418 | heat shock protein,HSP |
14 | LOC_Os01g69220 | exostosin family domain containing protein |
15 | LOC_Os01g38970 | carbamoyl-pHospHate synthase large chain |
16 | LOC_Os03g57790 | 泛素结合酶,UBC |
17 | LOC_Os08g41990 | Aminotransferase |
18 | LOC_Os10g08550 | 2-磷酸甘油酸水解酶,enolase |
19 | LOC_Os06g08170 | MFS transporter |
20 | LOC_Os11g25980 | phospholipid-transporting ATPase2 |
21 | LOC_Os08g44280 | L-异天冬氨酸甲基转移酶(OsPIMT1) |
22 | LOC_Os02g33500 | threonyl-tRNA synthetase,putative |
组合 | 质粒1 | 质粒2 | 目的 | 缺陷培养基 | 生长状态 |
---|---|---|---|---|---|
1 | pOst-Nub1 | pBT3-N | 阳性对照 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ √ |
2 | pPR3-N-1 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ × |
3 | pPR3-N-2 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ × |
… | … | … | … | … | … |
16 | pPR3-N-15 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ √ |
… | … | … | … | … | … |
23 | pPR3-N-22 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ × |
表5 膜系统筛选出的候选互作蛋白的自激活检测
组合 | 质粒1 | 质粒2 | 目的 | 缺陷培养基 | 生长状态 |
---|---|---|---|---|---|
1 | pOst-Nub1 | pBT3-N | 阳性对照 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ √ |
2 | pPR3-N-1 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ × |
3 | pPR3-N-2 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ × |
… | … | … | … | … | … |
16 | pPR3-N-15 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ √ |
… | … | … | … | … | … |
23 | pPR3-N-22 | pBT3-N | 自激活检测 | SD-Lue-Trp SD-Lue-Trp-His-Ade | √ × |
编号 | 基因ID | 功能预测 |
---|---|---|
1 | LOC_Os10g28254 | Expressed protein |
2 | LOC_Os03g15570 | Protein kinase-like |
3 | LOC_Os12g08760 | Carboxyvinyl-carboxyphosphonate Phosphorylmutase |
4 | LOC_Os 01g64660 | Fructose-1,6-bisphosphatase |
5 | LOC_Os 12g18110 | Lysosomal Cystine Transporter family protein |
6 | LOC_Os 05g45950 | VDAC(outer mitochondrial membrane porin) |
7 | LOC_Os 02g33820 | Abscisic stress-ripening |
表6 核体系下筛选的LCD候选互作蛋白
编号 | 基因ID | 功能预测 |
---|---|---|
1 | LOC_Os10g28254 | Expressed protein |
2 | LOC_Os03g15570 | Protein kinase-like |
3 | LOC_Os12g08760 | Carboxyvinyl-carboxyphosphonate Phosphorylmutase |
4 | LOC_Os 01g64660 | Fructose-1,6-bisphosphatase |
5 | LOC_Os 12g18110 | Lysosomal Cystine Transporter family protein |
6 | LOC_Os 05g45950 | VDAC(outer mitochondrial membrane porin) |
7 | LOC_Os 02g33820 | Abscisic stress-ripening |
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