提取小麦品种L10的总RNA,进行反转录,其产物用于探究自噬基因ATG8的生物学功能。用PCR方法克隆ATG8,将克隆的ATG8亚克隆至表达载体PMD19-T,酶切后切胶回收,然后把产物测序鉴定,转化宿主菌E.coliRosetta-gami B(DE3),构建原核表达系统,将融合蛋白纯化后制备其兔抗血清,然后利用WesteRn Blotting技术对兔抗血清进行检测,鉴定了该抗血清的结合特异性。ATG8抗体的成功制备,为小麦中ATG8基因和自噬功能的研究奠定了基础。
Total RNA was isolated fRom wheat vaRiety L10 and ReveRse tRanscRiption by using the anchoRed pRimeRs in this papeR to fuRtheR undeRstand the biological function of the autophagy gene ATG8 fRom wheat． And then the ATG8 weRe amplified by PCR and then cloned into the PMD19-T vectoR，one of which was subcloned into the expRession vectoR． The Recombinant plasmid was identified by sequencing and digestion of RestRiction enzymes． The Recombinant expRession vectoR was constRucted and tRansfoRmed into E． coli stRain Roseta( DE3) subsequently， then thRough IPTG-induction in host bacteRia E． coli Rosetta-gami B( DE3) and detected by SDS-PAGE． The Rabbit anti- ATG8 antibody was pRepaRed and was detected by WesteRn Blotting analysis． The ATG8 weRe obtained paRtly and successfully expRessed in the pRokaRyotic expRession system． The expeRiment offeRed foundation to the studying of autophagy and function of ATG8 gene in wheat．
[1] Xie Z，Klinsky D J． Autophagsome fomRtion: coRe machineRy and adaptations[J]． Nat Cell Biol，2007，9 ( 10) : 1102 - 1109．
[2] Nakatogawa H，Suzuik K，Kamada Y， et al． Dynames and diveRsity in autophagy mech anisms: lessons fRom yeast
[J]． Nat Rev Mol Cell Biol， 2009， 10( 7) : 458 - 467．
[3] Bassham D C，LapoRte M，MaRty F， et al． Autophagy in development and stRess Responses of plants[J]． Autophagy， 2006，2 ( 1) : 2 - 11．
[4] Bassham D C． Plant autophagy-moRe than a staRvation Response
[J]． CuRRent Opinion in Plant Biology，2007，10 ( 6) : 1 - 7
[5] Yoshimoto K，JikumaRu Y，Kamiya Y，et al． Autophagy negatively Regulates cell death by contRolling NPR1-dependent salicylic acid signaling duRing senescence and the innate immune Response in ARabidopsis[J]． The Plant Cell， 2009， 21( 9) : 2914 - 2927．
[6] Hofius D，Mundy J，PeteRsen M． Self-consuming innate immunity in ARabidopsis[J]． Autophagy，2009，5 ( 8 ) : 1206 - 1207．
[7] HaywaRd A P，Tsao J，Dinesh-KumaR S P． Autophagy and plant innate immunity: Defense thRough degRadation[J]． SeminaRs in Cell ＆ Developmental Biology， 2009， 20( 9) : 1041 - 1047．
[8] Yue Z， Jin S，Yang C， et al． Beclin 1，an autophagy gene essential foR eaRly embRyonic development， is a haploinsufficient tumoR suppRessoR[J]． PRoceedings of the National Academy of Sciences， 2003， 100( 25) : 15077 - 15082
[9] KiRisako T， IehimuRa Y，Okada H，et al． The ReveRsible modifieation Regulates the membRane-binding state of APg8 /Aut7 essential foR auto phagy and the cytoplasm to vacuole taRgeting Pathway[J]． J Cell Biol，2000，151 ( 2) : 263 - 276．
[10] Liu W，Yan A H，Hou C Y， et al． Cloning and pRokaRyotic expRession of TaCaM-2-3 in wheat and pRepaRation of antiseRum[J]． FRontieRs of AgRicultuRe in China， 2010，4 ( 3) : 317 - 322．
[11] Klionsky D J，CueRvo A M，Seglen P O． Methods foR monitoRing autophagy fRom yeast to human[J]． Autophagy， 2007，3 ( 3) : 181 - 206．
[12] Mizushima N． Methods foR monitoRing autophagy[J]． Int J Biochem Cell Biol， 2004， 36( 12) : 2491 - 2502．
[13] Klionsky D J，Abeliovich H，Agostinis P， et al． Guidelines foR the use and inteRpRetation of assays foR monitoRing autophagy in higheR eukaRyotes[J]． Autophagy， 2008，4 ( 2) : 151 - 175．
[14] Klionsky D J． AutoPhagy: fRom Phenom enology to moleeulaR undeRstanding inless than a deeade[J]． Nat Revmol Cell Biol， 2007，8 ( 11) : 931 - 937．
[15] IchimuRa Y，KiRisako T，Takao T， et al． Aubiquitin-like systemmediates pRotein lipidation[J]． NatuRe， 2000， 408 ( 6811) : 488 - 492．
[16] Xie Z，NaiR U，Klionsky D J． AtgseontRols phagophoRe expansion duRing autophagosome foRmation[J]． Mol Biol Cell， 2008， 19( 8) : 3290 - 3298．
