梨幼果FWL1膜系统酵母双杂交三框cDNA文库构建及互作蛋白的筛选

doi:10.6048/j.issn.1001-4330.2022.08.008

摘要/Abstract

摘要：

【目的】筛选与FWL1互作的蛋白,为研究FWL1基因调节果实大小机理提供依据。【方法】以杜梨、库尔勒香梨、鸭梨、早美香为材料,提取4个梨品种果实细胞分裂关键时期的果肉组织混样RNA。构建梨幼果FWL1膜系统酵母双杂交三框cDNA文库,并鉴定文库质量。构建诱饵载体并转化酵母菌,筛选与FWL1互作的蛋白,对初步筛选出的阳性酵母克隆进行测序并在NCBI中进行Blast比对,确定候选互作蛋白。【结果】文库库容约为3×10⁷ CFU,大于1×10⁷ CFU,平均插入片段大于1 000 bp,阳性率为≥98%。诱饵载体无自激活功能,利用共转化方法,筛选出272个与FWL1互作的蛋白质。【结论】梨幼果膜系统酵母双杂交三框cDNA文库符合酵母双杂文库的基本条件,适用于后期筛选相互作用的蛋白。筛选出的272个互作蛋白在果实发育过程中表达不同,其中collagen and calcium-binding EGF domain-containing protein 1和metallothionein-like protein可能与梨果实大小发育有关。

关键词: 梨; 果实大小; fw2.2; 三框cDNA文库; 互作蛋白筛选

Abstract:

【Objective】 Screening of proteins that interact with FWL1 provides a basis of in-depth exploration of the mechanism of FWL1 gene regulation of fruit size. 【Methods】 Using ‘Duli’ pear, Korla fragrant pear, and Yali pear and Zaomeixiang were used as materials, to extract the mixed RNA of pulp tissue in the critical period of fruit cell division of four pear varieties was extracted. And then a yeast two-hybrid triple-frame cDNA library of pear young fruit FWL1 membrane system was constructed, and the quality of the library was identified. After that, The the bait vector was constructed and transformed into yeast, and the proteins that interacted with FWL1 were screened. The positive yeast clones that were initially screened were sequenced and Blast alignment was performed in NCBI to determine the candidate interacting proteins. 【Results】 The library capacity is was about 3×10⁷ CFU, greater than 1×10⁷ CFU, the average insert fragment is was greater than 1,000 bp, and the positive rate is was ≧98%. After testing the bait vector had no self-activating function, and 272 proteins interacting with FWL1 were screened out by co-transformation method. 【Conclusion】 After testing, the yeast two-hybrid three-frame cDNA library of the pear young fruit membrane system meets the basic conditions of the yeast two-hybrid library and is suitable for later screening of interacting proteins. The screened 272 interacting proteins were are expressed differently during fruit development, among which collagen and calcium-binding EGF domain-containing protein 1 and metallothionein-like protein may be related to pear fruit size development.

Key words: pear; fruit size; fruit weight 2.2; construction of three-frame cDNA library; screening of interacting proteins

中图分类号:

S661.2

赛静忆, 温玥, 郝志超, 田嘉. 梨幼果FWL1膜系统酵母双杂交三框cDNA文库构建及互作蛋白的筛选[J]. 新疆农业科学, 2022, 59(8): 1877-1888.

SAI Jingyi, WEN Yue, HAO Zhichao, TIAN Jia. Construction of Yeast Two-Hybrid Three-Frame cDNA Library and Screening of Interacting Proteins in FWL1 Membrane System of Pear Young Fruit[J]. Xinjiang Agricultural Sciences, 2022, 59(8): 1877-1888.

图/表 12

参考文献 39

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试剂 Reagent	单位 Unit
First-Strand cDNA	2
Deionized H₂O	80
10× Advantage 2 PCR Buffer	10
50× dNTP Mix	2
M1 PCR Primer	4
50× Advantage 2 Polymerase Mix	2

试剂 Reagent	单位 Unit
First-Strand cDNA	2
Deionized H₂O	80
10× Advantage 2 PCR Buffer	10
50× dNTP Mix	2
M1 PCR Primer	4
50× Advantage 2 Polymerase Mix	2

试剂 Reagent	第1次扩增（μL） First amplification/μL	第2次扩增（μL） Second amplification/μL
cDNA(DSN处理稀释后)
cDNA (after DSN treatment and dilution)	1	0
cDNA(稀释后) cDNA (after dilution)	0	2
纯化水purified water	40.5	80
10× Advantage 2 PCR Buffer	5	10
50× dNTP Mix (10mM of each)	1	2
Evrogen PCR primer M1	1.5	0
Evrogen PCR primer M2	0	4
50× Advantage 2 Polymerase Mix	1	2

试剂 Reagent	第1次扩增（μL） First amplification/μL	第2次扩增（μL） Second amplification/μL
cDNA(DSN处理稀释后)
cDNA (after DSN treatment and dilution)	1	0
cDNA(稀释后) cDNA (after dilution)	0	2
纯化水purified water	40.5	80
10× Advantage 2 PCR Buffer	5	10
50× dNTP Mix (10mM of each)	1	2
Evrogen PCR primer M1	1.5	0
Evrogen PCR primer M2	0	4
50× Advantage 2 Polymerase Mix	1	2

引物名称 Primers	引物序列（5’→3’） Primers sequence
FWL1-F（pBT3-N）	GAATTCCTGCAGGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-N）	TACTTACCATGGGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC
FWL1-F（pBT3-C）	CACTAATCTAGACGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-C）	ATGGAGGCCTTTGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC
FWL1-F（pBT3-SUC）	AATATCTGCAATGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-SUC）	ATTCCTGCAGATGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC
FWL1-F（pBT3-STE）	TATTTTATGTAATGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-STE）	ATTCCTGCAGATGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC