Bhide Amey J, Channale Sonal M, Yadav Yashpal, Bhattacharjee Kabita, Pawar Pankaj K, Maheshwari V L, Gupta Vidya S, Ramasamy Sureshkumar, Giri Ashok P
Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411 008, India.
Department of Biochemistry, Shivaji University, Kolhapur, 416 004, India.
Plant Mol Biol. 2017 Jun;94(3):319-332. doi: 10.1007/s11103-017-0609-5. Epub 2017 Apr 12.
The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3'UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.
报道了来自皱果苋（AAI）的最小的32个氨基酸的α-淀粉酶抑制剂。成功克隆了编码26个氨基酸（aa）信号肽、随后是43个氨基酸区域和先前鉴定的32个氨基酸肽的前体蛋白（AhAI）的完整基因。AhAI中存在已知α-淀粉酶抑制剂中保守的三个半胱氨酸残基和一个二硫键。在皱果苋的近缘种即圆锥苋、牛膝和青葙中发现了相同的基因组和开放阅读框。有趣的是，AhAI的3'UTR在这些物种中有所不同。AhAI在皱果苋花序中的表达最高；然而，在种子中未检测到。我们推测在叶片和花序中表达的抑制剂可能会转运到种子中。亚细胞定位研究清楚地表明AhAI信号肽参与细胞外分泌。全长rAhAI对来自人、昆虫、真菌和细菌的α-淀粉酶表现出不同的抑制作用。特别是，棉铃虫（鳞翅目）的α-淀粉酶不受AhAI抑制，而赤拟谷盗和绿豆象（鞘翅目）的α-淀粉酶则被完全抑制。AhAI的分子对接显示，与绿豆象α-淀粉酶相比，它与赤拟谷盗α-淀粉酶活性位点残基的相互作用更紧密，这可能是它们IC差异的原因。以rAhAI为食后，绿豆象的正常生长、发育和成虫羽化受到阻碍。总之，AhAI影响绿豆象生长的能力证明了它作为一种高效生物防治剂的潜力，特别是对储粮害虫。
