摘要
通过对抗性和敏感棉铃虫中肠氨肽酶的克隆和测序,鉴定了氨肽酶基因家族的2个成员:Haapn3和Haapn4,其cDNA序列具有3053和2862个核苷酸,分别具有3045和2853bp的开放阅读框,编码产生1014和951个氨基酸的蛋白质。其推定氨基酸序列均具有锌结合模体HEXXH和所有锌依赖氨肽酶共有的序列GAMEN。N 末端分别具有18和20个氨基酸的疏水性信号肽,C 末端均具有21个氨基酸的GPI添加信号肽。比对抗性和敏感棉铃虫cDNA的开放阅读框,在Haapn3的开放阅读框中,抗性品系的cDNA具有25个核苷酸的不同,其中9个碱基替代造成氨基酸的变化:苏氨酸189→天冬酰胺、甘氨酸229→天冬氨酸、赖氨酸231→精氨酸、谷氨酸250→丙氨酸、异亮氨酸275→缬氨酸、天冬酰胺281→天冬氨酸、酪氨酸302→组氨酸、缬氨酸648→异亮氨酸、脯氨酸975→谷氨酰胺,其它16个碱基替代为无义突变。在Haapn4的开放阅读框中,有15个点突变,其中8个突变为无义突变,其它7个导致了氨基酸的改变,即赖氨酸4→天冬酰胺、组氨酸162→酪氨酸、脯氨酸185→丝氨酸、谷氨酸297→甘氨酸、苯丙氨酸385→丝氨酸、甲硫氨酸450→异亮氨酸、缬氨酸630→异亮氨酸。本文报道的氨肽酶4个cDNA序列已提交GenBank,AY279536和AY279537分别是抗性和敏感品系的Haapn3。
Loss of binding-site on receptor or reducing of binding affinity between receptor and toxin has been explained as a reason for insect resistance to Bt endotoxin. In many lepidopteran insects aminopeptidase N from larvae midgut has been reported to be a receptor of Cry1A toxin from Bacillus thuringiensis. cDNAs of aminopeptidase N from the fifth instar larvae midgut of Helicoverpa armigera Hübner susceptible (YCS strain) and resistant (YCR strain) to Bt transgenic cotton expressing Cry1Ac toxin are cloned and sequenced. The cDNA sequences were aligned and analyzed with BioEdit software, and the deduced amino acid sequences were analyzed by Signal P and Big-πProdictor programs. Two members Haapn3 and Haapn4 in aminopeptidase N family were identified, and had been deposited in the GenBank database (accession number AY279536 and AY279537 for Haapn3 from resistant and susceptible strain, AF511038 and AY279534 for Haapn4 from resistant and susceptible strain). The cDNAs of Haapn3 and Haapn4 have 3053 and 2862 nucleotides with a 3045bp and 2853bp open reading frame that encode a 1014 and 951 amino acids respectively. Both the two proteins share the consensus zinc binding/gluzincin motif HEXXH and GAMEN sequence common to gluzincin aminopeptidases. And hydrophobic signal sequence with 18-20 amino acids in N-terminal region and a GPI anchor site in C-terminal region are present in these two aminopeptidases. Alignment and comparison of cDNA sequences from Bt-susceptible and Bt-resistant cotton bollworm revealed some nucleotide differences in the open reading frames. cDNA of Haapn3 from resistant strain has 25 nucleotide point mutations, in which nine lead to amino acid changes: Thr_(189)→Asn、Gly_(229)→Asp、Lys_(231)→Arg、Glu_(250)→Ala、Ile_(275)→Val、Asn_(281)→Asp、Tyr_(302)→His、Val_(648)→Ile、Pro_(975)→Gln. In the open reading frame of Haapn4 there are 15 point mutations, eight of them are silent, the other seven result in amino acid changes in putative protein sequence of aminopeptidases: Lys_4→Asn、His_(162)→Tyr、Pro_(185)→Ser、Glu_( 297)→Gly、Phe_(385)→Ser、Met_(450)→Ile、Val_( 630)→Ile.
出处
《棉花学报》
CSCD
北大核心
2004年第1期13-20,共8页
Cotton Science
基金
教育部博士点基金(2000030703)
国家自然科学基金(30270889)
