芝麻與愛玉半胱胺酸水解酵素抑制劑對四紋豆象抑制效應之研究

Study on the Inhibitory Effects of Phytocystatins from Sesame and Jelly Fig Against the Cowpea Weevil Callosobruchus maculatus

方納多(Ito Fernando)

指導教授 ： 徐志宏 ； Retno Dyah Puspitarini

國立屏東科技大學/農學院/生物科技系/碩士(2019年)

https://doi.org/10.6346/NPUST201900388

摘要

已知植物抑制素作為植物中的天然防禦機制，以對抗食草害蟲的侵襲。近年來，表達胱抑素的轉基因作物的發展受到極大關注。然而，鑑於幾種昆蟲克服障礙的能力，不斷需要新的遺傳資源來擴大轉基因植物的有效性和增加其耐久性。在這裡，從芝麻（SiCYS）和果凍無花果（FaCYS）中獲得了編碼植物抑素的cDNA，並將其構建為pET載體系統作為其標記的蛋白質並在 大腸桿菌BL21（DE3)。首先進行木瓜蛋白酶抑制測定以證實純化的重組植物生成抑制素的功能，然後對在Callosobruchus maculatus幼蟲中發現的中腸酶進行體外抑制測定。使用人工種子系統進行飼養測定以闡明兩種抑製劑對C. maculatus的整體生物學的能力。SiCYS和FaCYS完全抑制了木瓜蛋白酶的活性，並顯著抑制了C. maculatus幼蟲的中腸酶蛋白水解活性，分別達到~75和~55％。飼餵試驗表明，兩種抑製劑均未能對昆蟲幼蟲造成明顯的死亡率。然而，當加入高劑量的SiCYS時，獲得了顯著的延遲對
C. maculatus發育期的影響。顯然，成人體重和豆類重量損失在治療組和對照組之間沒有差異，表明抑製劑對C. maculatus幼蟲晚期沒有拒食作用。考慮到這一顯著的結果，可以考慮將SiCYS用作有希望的候選轉基因基因用於未來的研究。

關鍵字

植物蛋白酶 ； 轉基因植物 ； 飼養試驗 ； 人工種子系統

並列摘要

Phytocystatins have been known to act as a natural defense mechanism in plants to combat herbivorous insect pests attack. In recent years the development of cystatin expressing transgenic crops is of great interest. However a new genetic resources are much needed for broadening the effectiveness and increasing durability of transgenic plants given the ability of several insects to overcome the barriers. Here a cDNA encoding phytocystatins had been obtained from sesame (SiCYS) and jelly fig (FaCYS) and were constructed into pET vector system as his-tagged protein and produced in
E. coli BL21(DE3). Initially, papain inhibitory assays were conducted to confirm the functionality of the purified recombinant phytocystatins, followed by in vitro inhibitory assay against midgut enzymes found in Callosobruchus maculatus larvae. A feeding assay using artificial seed system were carried out to elucidate the ability of both inhibitors on the overall biology of C. maculatus. SiCYS and FaCYS completely inhibited the papain activity, and dramatically suppressed the midgut enzymes proteolytic activity of C. maculatus larvae up to ~75 and ~55%, respectively. Feeding assay revealed that both inhibitors were failed to cause noticeable mortality on the insect larvae. Nevertheless, a pronounced retardation effects on the developmental period of C. maculatus were obtained when high dose of SiCYS was incorporated. Apparently, the adults body weight and beans weight losses didn’t differ amongst the treatment and control group, indicating no antifeedant effects of the inhibitors on the late stage of C. maculatus larvae. Taking into account of this prominent results, the utilization of SiCYS as a promising candidate transgenic gene might be considered for future studies.

並列關鍵字

phytocystatins ； transgenic plants ； feeding assay ； artificial seed system

參考文獻

REFERENCES

Abe K., Y. Emori, H. Kondo, S. Arai, K. Suzuki. 1988. The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J. Biol. Chem. 263(16): 7655-7659.

Aguiar J.M., O.L., Franco, D.J. Rigden, C. Bloch, A. Monteiro, V.M. Flores, T. Jacinto, J. Xavier-Filho, A.E.A. Oliveira, M.F., Grossi-de-Sa, K.V.S. Fernandes. 2006. Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests. Proteins: Struc. Func. Bioinform. 63(6): 662-670.

Ahn J.E., M.R. Lovingshimer, R.A. Salzman, J.K. Presnail, A.L. Lu, H. Koiwa, K. Zhu-Salzman. 2007. Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes. Insect Mol. Biol. 16(3): 295-304.

Amirhusin B., R.E. Shade, H. Koiwa, P.M. Hasegawa, R.A. Bressan, L.L. Murdock, K. Zhu-Salzman. 2007. Protease inhibitors from several classes work synergistically against Callosobruchus maculatus. J. Insect Physiol. 53: 734-740.