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Sequence and Expression of MRJPs cDNA from Apis Cerana Cerana

Author: XingLiPing
Tutor: ZhangChuanXi；ShenLiRong
School: Zhejiang University
Course: Agricultural Entomology and Pest Control
Keywords: Apis cerana cerana royal jelly prokaryotic expression eukaryotic expression sequence analysis apisimin MRJP1 MRJP2 MRJP3 MRJP3a
CLC: S893
Type: Master's thesis
Year: 2006
Downloads: 93
Quote: 1
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Abstract

The Chinese honeybee Apis cerana cerana is specially distributed in most parts of China, especially in the Mountains areas of South China, and is adapted to the geographic and climate condition. About two to three millions of colonies were bred in China per years.The development of a honeybee larva depends on a complex secretion of the hypopharyngeal and mandibular glands of nurse workers. The complex secretion is called royal jelly. The composition of royal jelly (RJ) varies with seasonal and regional conditions. The RJ contains average moisture of 60-70%, crude protein 12-15%, total sugar 10-16%, lipids 3-6%, small quantities of vitamins, salts and free amino acids.It has been reported that RJ has several pharmacological activities, including vasodilative and hypotensive activities, increase in growth rate of chick embryos, disinfectant action, antitumor activity, anti-inflammatory activity, antihypertensive activity, antifatigue activity and antiallergy activity.A substantial part of RJ is made of proteins, which are form about 50% of the dry mass of RJ. RJ proteins belong to one protein family. Nine members of the major royal jelly proteins (MRJPs) (49 - 87 kDa) family have been identified up to date. Proteins MRJP1, MRJP2, MRJP3s, MRJP4, MRJP5s represent about 82 % of total protein content of RJ. It is generally accepted that they play a role as a source of essential amino acids and nitrogen in the nutrition of honeybee larvae.In this report, we constructed a cDNA library from 8-day-old nurse honeybee heads of A. cerana cerana, screened out the clones containing the cDNA encoding MRJP1, MRJP2, MRJP3, and MRJP3a and apisimin. Apisimin gene was expressed in E. coli and MRJP2 expressed in insect cells.Sequence Analysis of MRJP-1, MRJP2, MRJP3, and MRJP3a from Apis cerana ceranaA cDNA library was constructed from eight-day-old worker heads of Apis cerana cerana. The clone of MRJP1, MRJP2, MRJP3, and MRJP3a was screened out. The sequence of AccMRJPl cDNA was 1451 bp long and included an open reading frame (ORF) of 1302 nucleotides encoding a protein of 433 amino acids. AccMRJP1 was highly similar (93%and 90% identity) to A. cerana indica at the nucleotide and protein level. The AccMRJP1 had 99% similarity with A. mellifera in nucleotide and amino acid sequences .The sequence of AccMRJP2 cDNA was 1652 bp long and included an open reading frame (ORF) of 1392 nucleotides encoding a protein of 463 amino acids.AccMRJP2 was highly similar (89%and 84% identity) to A. mellifera at the nucleotide and protein level. The AccMRJPl had 99% similarity with A. cerana indica in nucleotide andamino acid sequences .The sequence of AccMRJP3 cDNA was 2062 bp long and included an open reading frame (ORF) of 1827 nucleotides encoding a protein of 608 amino acids.AccMRJP3 had 82%, 99% , 77%, 73% to A. mellifera , A. cerana indica ^ A.dorsata > A.florea at the protein level. MJRP3a cDNA was 2150bp long and included an open reading frame (ORP) of 1719 nucleotides encoding a protein of 572 amino acids.AccMRJP3a had 76%^ 91% , 75%^ 72% toA. mellifera , A. ’cerana indica , A.dorsata, A.florea at the protein level. MRJP3 exhibts a size polymorphism. MRJP3a is variant of MRJP3 .N-terminal sequence is highly homologous. A repetitive region was found at the C-terminal region of the AccMRJP3 ORF and AccMRJP3 ORF.Sequence Analysis of Apisimin cDNA from Apis cerana cerana and its Expression in E. coliThe clone of apisimin was screened out. The fragment was 379 nt in length, containing an open-reading frame(ORF) encoding 78 amino acids. The Apisimin of Apis cerana cerana shared 100 % and 95% homology with Apis mellifera and A.cerana indica in nucleotide. The coding region of the matured peptide was sub-cloned into the prokaryotic expression vector pGEX-4T-2. The vector was then transformed into E. coli BL21 (DE3) for expression.. Analysis of the result of SDS-PA GE showed that the expression products contained a band of protein of about 31.9kD. The SDS-PAGE showed that most of recombinant protein was soluble. The apisimin was obtained by purification through Glutathione Sepharose 4B affinity chromatography.Cloning and expression of MRJP2 in insect cellMRJP2 gene was inserted into pFastBacHTa to constructed pFastBacHTa-MRJP2, The recombinant donor plasmids were transformed to DH10 competent cells and constructed the recombinant Bacmid, Bacmid-MRJP2. Bacmid-MRJP2 was’then transfected into insect cells for expression..SDS-PAGE analysis showed the expression product of MRJP2 gene was 50kD, consistent with the predicted molecular weight, suggesting that no most post-translation modification occurred in this protein. SDS-PAGE and thin layer scanning methods showed the expressed MRJP2 reached to 21.3 % of the total proteins.

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