摘要
黄花石蒜凝集素(Lycoris chinensis Agglutinin, LCA)是来源于石蒜科的具有多重生物学活性的甘露糖结合蛋白,属于单子叶甘露糖结合凝集素。这类凝集素由于具有独特的糖结合特异性、逆转录病毒抑制活性和保护农作物免受昆虫和线虫攻击而引起人们浓厚的兴趣。本文采用同源克隆的方法,通过对黄花石蒜球茎RNA的提取,反转录成cDNA,从Genbank上比较已登录的石蒜科凝集素的序列,找出其保守序列,并结合软件设计了简并引物,进行3’RACE末端快速扩增,得到了3’端的部分序列(序列号为AY690318),序列长为516bp,然后根据已得到的序列设计特异性引物,应用在cDNA的3’端随机添加若干PolyA的方法,在cDNA模板3’端加上PolyA,再以此为模板进行PCR,从而克隆得到了黄花石蒜5’端的部分序列(序列号为AY763111),序列长为411bp,将3’与5’序列合并,就能获得cDNA全长序列(序列号为AY763112)。
以获得的全长cDNA序列和推导出的氨基酸序列与石蒜科其他植物凝集素在Genbank中的Blast上进行比较,可知黄花石蒜的全长cDNA序列由683个核苷酸组成,开放阅读框长486bp,编码162个氨基酸,起始密码子位于第37-39bp,终止密码子位于在523-525bp处,在起始密码子上游有一个由36个碱基组成的5’非编码区:在终止密码子下游有一个由141个碱基组成的3’非编码区,包括两个加poly(A)信号和一段真核生物mRNA所特有的poly(A)序列。通过序列的测定,发现LCA基因均编码前体蛋白:含有信号肽序列、成熟蛋白序列和C-末端剪切序列。该成熟蛋白由109个氨基酸组成,分子量为12.1KD。
Lycoris chinensis Agglutinin (LCA) from Amaryllidaceae species is a mannose-binding protein with various biological activities. It belongs to the monocot mannose-binding lectin (MBL). The lectins have received a lot of attention in different scientific disciplines because of their unique and exclusive specificity towards mannose, their retrovirus inhibitory activity and toxicity to insects. The total RNA of Lycoris chinensis was extracted, and then reverse transcript into cDNA. A primer was designed based on the conserved regions of other MBL plant's agglutinin through homology alignment. Using the RT-PCR, 3'RACE (rapid amplification of cDNA ends) technique, a DNA fragments was first cloned from Lycoris chinensis by performing PCR that used Lycoris chinensis cDNA genome as the template. It has 516 nucleotides (accession number in Genbank: AY690318). Then another two primers were designed according to the sequence of 3'ends, one was used to reverse transcript the RNA into cDNA, poly A were added to the 3'ends of cDNA with the function of TdT (Terminal Deoxynucleotidyl Transferase), using the 5'RACE technique the fragment of 411 nucleotides was cloned (accession number in GenBank: AY763111). The two partially overlapping cDNA fragments were assembled a full-length cDNA sequence of Lycoris chinensis (accession number in Genbank: AY763112).The full-length cDNA had 683bp, and the sequence encoded an open reading frame of 162 amino acids. The start codon was at 37-39 bp and the stop codon was
at 523-525 bp. The sequence also contained 5'nontranslated region with 36bp and 3' nontranslated region with 141bp. The result showed that LCA gene encoded a protein precursor with a signal peptide^ mature protein and C-terminal cleavage amino acids sequence by the analysis in the Blast of Genbank. The mature protein included 109 amino acids residues and the molecular weight is 12.1 KD. The mature protein sequence showed the identity to those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiate agglutinin, Clivia miniata agglutinin, 7ephranth.es grandiflora agglutinin, Amaryllis vittata agglutinin, Zephyranthes Candida Herb agglutinin respectively are 82.6% > 83.5% ^ 77.3% > 83.5% ? 80.9%, 79.^ 72.7 % Blocks'analysis revealed that the deduced amino acid sequence of LCA had three functional domains specific for lectin and three sugar-binding boxes (QDNY). In the mature protein, there were 34.9% hydrophobic amino acids, 46.8% hydrophilic amino acids, 8.3% basic amino acids and 10.1% acidic amino acids. The signal peptide of LCA had 23 amino acids, included a hydrophobic core of 9 amino acids. The cloning of this gene established an important base to the study of LCA gene structure, the cleavage mechanism, the mechanism of expression and regulation, the relationship of structure and function of LCA.
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