杞�(zhu菐n)鍩哄洜娓�(c猫)瑭︾洅
鐢�(ch菐n)鍝佸瀷铏�(h脿o)锛欵PSPSCP4鐢�(ch菐n)鍝佸児(ji脿)鏍硷細鍦ㄧ窔瑭㈠児(ji脿)
杞�(zhu菐n)鍩哄洜娓�(c猫)瑭︾洅/EPSPSCP4杞�(zhu菐n)鍩哄洜瑭﹀姂鐩�鏄敤鐗圭暟鎬ф姉 Cry1Ab/1Ac 鍠姉鍖呰鐨勫井瀛旀澘鍜岄叾妯�(bi膩o)瑷樻姉 Cry1Ab/1Ac 鍠姉锛岄洐鎶楅珨澶惧績娉曟娓�(c猫) Cry1Ab/1Ac 鍩哄洜琛ㄩ仈(d谩)鐢�(ch菐n)鐗�锛屽彲浠ラ珮闈堟晱搴﹀拰鐗圭暟鎬х殑妾㈡脯(c猫)杞�(zhu菐n)鍩哄洜妞嶇墿涓殑 Bt-Cry1Ab/1Ac 铔嬬櫧銆�
銆� 瑭﹀姂鐩掑師鐞嗐€�
鏈搧绯荤敤鐗圭暟鎬ф姉 Cry1Ab/1Ac 鍠姉鍖呰鐨勫井瀛旀澘鍜岄叾妯�(bi膩o)瑷樻姉 Cry1Ab/1Ac 鍠姉锛岄洐鎶楅珨澶惧績娉曟娓�(c猫) Cry1Ab/1Ac 鍩哄洜琛ㄩ仈(d谩)鐢�(ch菐n)鐗�锛屽彲浠ラ珮闈堟晱搴﹀拰鐗圭暟鎬х殑妾㈡脯(c猫)杞�(zhu菐n)鍩哄洜妞嶇墿涓殑 Bt-Cry1Ab/1Ac 铔嬬櫧銆�
銆愯│鍔戠洅绲勪唤锛堣(gu墨)鏍硷級銆�
闋�(y霉)鍖呰鏉匡紙鎶� Cry1Ab/1Ac 鍠姉锛� 12 瀛�×8
Cry1Ab/1Ac 閰舵(bi膩o)鎶楅珨 12 ml×1 鐡�
20×婵冪府娲楁粚娑� 30 ml×1 鐡�
5×鎶芥彁娑�/绋€閲嬬珐娌栨恫 30 ml×1 鐡�
搴曠墿娑� A 7 ml×1 鐡�
搴曠墿娑� B 7 ml×1 鐡�
绲傛娑� 7 ml×1 鐡�
浣跨敤瑾槑鏇� 1 浠�
0ppb 闄版€у皪(du矛)鐓� 1ml
4 鍊�(g猫)涓嶅悓婵冨害鐨勬(bi膩o)妯o細
0.5 ppb 1.0 ppb 2.0 ppb 4.0 ppb 鍚� 1ml
銆愯│鍔戞簴(zh菙n)鍌欍€�
瑭﹀姂鐩掍腑鐨� 20×婵冪府娲楁粚娑� 30 ml 鍔� 570 ml 婊呰弻 ddH2O 婧惰В绋€閲嬭嚦 1×娲楁粚娑�锛�5×鎶芥彁娑诧紙30 ml锛夊姞 120 ml ddH2O 绋€閲嬭嚦 1×鎶芥彁娑诧紝4鈩冨瓨鏀惧倷鐢�銆�
銆愭ǎ鍝佹簴(zh菙n)鍌欍€�
绋卞彇绱� 20 mg 钁夌墖锛屽姞 0.5 ml 1×鎶芥彁娑诧紙浣跨敤鍓� 30 min 鏀剧疆瀹ゆ韩锛夌爺纾ㄨ嚦鍕绘伎锛屽婧斁缃� 30 min锛屾脯(c猫)瀹氬墠鎶婃ǎ鍝佷笂娓呮恫绋€閲� 5-20 鍊�銆�
娓�(c猫)瀹氳帠骞插拰鑳氫钩涓殑 Bt 铔嬬櫧鏅�(sh铆)锛屾ǎ鍝佺ū鍙栫磩 40 mg锛屽姞 1 ml 1×鎶芥彁娑茬爺纾�銆�
銆怑LISA 鍙嶆噳(y墨ng)銆�
1. 渚濇鍔� 100 μl 闄版€у皪(du矛)鐓�锛屾(bi膩o)婧�(zh菙n)鍝侊紙4 鍊�(g猫)涓嶅悓婵冨害鐨勬(bi膩o)妯o細0.5 ppb锛�1.0 ppb锛�2.0 ppb, 4.0 ppb锛夊拰寰呮脯(c猫)妯e搧鑷� 96 瀛旀澘锛�
2. 鐢ㄥ皝鍙h啘钃嬩綇 96 瀛旀澘锛屽揩閫熸悥鍕�(d貌ng)骞虫澘娣峰嫽妯e搧锛�
3. 37鈩冩亽婧瀛佃偛 30 min锛�
4. 鍊掓帀 96 瀛旀澘涓恫楂旓紝姣忓瓟鍔犲叆 1×娲楁粚娑� 250 μl 娲楁粚寰瓟鏉�锛屾礂鏉� 5 娆★紝鐢ㄥ惛姘寸礄鎷嶅共锛�
5. 鎸夐爢搴忓湪姣忓€�(g猫)灏忓瓟涓姞鍏� 100 μl Cry1Ab/1Ac 閰舵(bi膩o)鎶楅珨锛岀劧鍚庨噸寰�(f霉)姝ラ 2锛�
6. 37 鈩冩亽婧瀛佃偛 30 min锛�
7. 鍊掓帀 96 瀛旀澘涓恫楂�锛屾瘡瀛斿姞鍏� 1×娲楁粚娑� 250 μl 娲楁粚寰瓟鏉匡紝娲楁澘 5 娆�锛岀敤鍚告按绱欐媿骞�锛�
8. 渚濇鍔犲叆 50 μl 搴曠墿 A 娑�锛�50 μl 搴曠墿 B 娑�锛岄噸寰�(f霉)姝ラ 2锛�
9. 瀹ゆ韩锛�20-25鈩冿級閬垮厜瀛佃偛 10 min锛屽姞 50 μl 绲傛娑�锛屽厖鍒嗘贩鍕荤祩姝㈠弽鎳�(y墨ng)锛�15min 浠ュ収(n猫i)鍦ㄩ叾妯�(bi膩o)鍎€涓婅畝鍙栫祼(ji茅)鏋溿€� 鍦� 450 nm 鐐轰富娉㈤暦锛�630 nm 鐐烘娉㈤暦锛堟垨鍠尝闀� 450 nm锛変笅锛岀敤绌虹櫧瀛旀牎闆�锛屽啀璁€鍙栧悇瀛� OD 鍊�銆�
銆愯綁(zhu菐n)鍩哄洜娓�(c猫)瑭︾洅浣跨敤娉ㄦ剰浜嬮爡(xi脿ng)銆�
1. 娓�(c猫)瀹氫腑闇€浣跨敤鐨勬墍鏈夎│鍔戝拰 96 瀛旀澘鎻愬墠 30 min 鏀剧疆瀹ゆ韩婧偛锛堝惁鍓囨渻(hu矛)褰遍熆瑭﹂(y脿n)婧�(zh菙n)纰烘€э級锛屾敞鎰�12 閫g蹇呴爤婧偛鍚庡啀寰炲钩鏉夸笂鍙栦笅锛屾湭浣跨敤瀹岀殑闋�(y霉)鍖呰鏉挎鎳�(y墨ng)缃簬鏈夊共鐕ュ姂鐨勫皝鍙h涓瘑灏佷繚瀛�锛�
2. 瑭﹀姂浣跨敤鍓嶆噳(y墨ng)鍏呭垎鎼栧嫽锛�
3. 鎼栧嫊(d貌ng)骞虫澘娣峰嫽鏅�(sh铆)娉ㄦ剰閬垮厤妯e搧涔嬮枔浜ゅ弶姹℃煋锛�
4. 浣跨敤娲楁粚娑叉紓娲楁檪(sh铆)锛屾礂婊屾恫闇€瑕佸~婊挎瘡鍊�(g猫)灏忓瓟锛岄€�(j矛n)琛屽厖鍒嗘紓娲�锛�
5. 鐮旂(濂界殑妯e搧濡傛灉涓嶈兘涓€娆″叏閮ㄦ脯(c猫)閲忥紝鍙互鏆檪(sh铆)鍦� 4 鈩冮伩鍏変繚瀛�锛屼絾鏄渶濂藉湪 24 h 鍏�(n猫i)娓�(c猫)瀹氾紱
6. 绲�(ji茅)鏋滃垽璁€璜�(q菒ng)?ji膩n)璇滅;?y墨ng)绲傛鍚� 15 min 鍏�(n猫i)瀹屾垚銆�
銆怋t 铔嬬櫧婵冨害瑷�(j矛)绠楀叕寮忋€�
Bt 铔嬬櫧婵冨害锛�μg/g锛�=
銆愬劜(ch菙)瀛樻浠跺強鏈夋晥鏈熴€�
瑭﹀姂鐩掑劜(ch菙)瀛樹簬 2-8鈩冿紝鍒囧嬁鍑嶅瓨锛涙湭闁嬪皝瑭﹀姂鐩掓湁鏁堟湡 12 鍊�(g猫)鏈�锛岄枊灏佸悗 3 鍊�(g猫)鏈堝収(n猫i)鐢ㄥ畬銆傦紙瑕嬪寘瑁濈洅涓婃(bi膩o)绨芥湁鏁堟湡锛�
銆愯綁(zhu菐n)鍩哄洜娓�(c猫)瑭︾洅鎶€琛�(sh霉)鍙冩暩(sh霉)銆�
鏈搧閬╃敤浜庣帀绫炽€佹鑺�銆佸ぇ璞嗕互鍙婂叾鍚勭ó杈�(n贸ng)浣滅墿涓綁(zhu菐n)鍩哄洜鎴愬垎鐨勫揩閫熷畾閲忔脯(c猫)瑭�銆�