Bt棉棉子中Bt蛋白定量检测方法建立及变化规律研究_徐宝梁

Abstract: Cot t onseeds, cot to nseed kernels and cot to nseed shells of 99B and 33B w ere st ored in a dry and dark place at room temperature. Samples w ere t aken every mo nt h and grounded. Ex t ract ions of cot t onseeds, kernels and shells w ere pr epar ed using t he method described in t he inst ruct ions o f the kit s. T he ex t ract ions w ere ser ially dilut ed w it h t he ext ract ion buff er s at the rat io o f 1 B1 ( volume t o volume) . Bt pr ot ein cont ent s of co tt onseed kernels w ere det erm ined using Envirolog ix kit f ol low ed the instr uction of t he kit . Optical densities o f t he ex t ract ion and the dilut ed ex t ract ions f rom cot t onseeds, kernels and shells at 450 nm and 630 nm w ere det ermined using Envirolog ix kit and Ag dia kit respect ively f ollow ed the inst ruct ions of t he kit s.
摘要: 应用 Enviro logix 试剂盒测定 99B、33B 棉子仁中 Bt 蛋白的含量, 分别为 4376. 4 ng # g-1
和 4321. 7 ng # g-1 ; 测定系列稀释的棉子仁提取液, Envirolo gix 试剂盒测得的 O D450 和 A g dia
试剂盒测得的 O D630间的相关性很好, 相关系数接近 0. 99; 而 A gdia 试剂盒测得的系列稀释液
Key words: Bt co tt onseed; Bt pro tein cont ent ; deg radation rule 没说从哪买的？
转基因棉花是我国目前唯一大规模种植的转 基因作物, 占棉花总种植面积的 50% 以上[ 1] 。已 有文献报道应用生物学和免疫学测定方法, 对棉 花植株各器官中 Bt 蛋白的含量进行测定, 测得不 同时期的植 株、不同时期和不同部 位的叶子、棉 铃、棉子中 Bt 蛋白的含量[ 2-5] , 确定了转基因棉花 植株中 Bt 蛋白的时空分布。有关转 Bt 基因棉花 的研究中, 棉子是一个非常重要的研究材料[ 6-8] , 但至今未见棉子贮存过程中 Bt 蛋白含量变化规 律的研 究 报 道。结 合 Bt 蛋 白 测 定 定 量、定 性 ELISA 试剂盒的特点, 建立了灵敏的 Bt 蛋白测 定定量方法, 据此研究了 DP 99B、新棉 33B 棉子、 棉子仁、棉子壳中 Bt 蛋白含量的变化规律。
T he Bt pro t ein cont ent s in 99B and 33B cot t onseed kernels w ere 4376. 4 ng # g-1and 4321. 7 ng # g-1 respect iv ely . T he O D450 of series of dilut ions of t he co tt o nseed ker nel ex t ract s measured using the Enviro logix kit w as linearly cor relat ed w it h t he OD630 det ect ed using Agdia kit , and the co rrelat ion coef ficient w as alm ost 0. 99. Furt herm ore, t he O D630 of t he dilutions w as also linearly corr elat ive wit h the content s o f Bt pro tein, and t he corr elat ion co ef ficient w as larg er t han 0. 99. T hus t he degradat ion rule of Bt pro tein in 99B and 33B cot t onseeds, kernels and shells in t he pro cess of 12 m onths st orage w ere det ermined using Ag dia kit , and the Bt prot ein cont ent s in t he cot t onseeds and kernels w ere half af t er t he st orag e, w hile the content s in t he shells w ere one f our th, bo th in 99B and 33B.
棉 花 学 报 Co tton Science 2006, 18( 1) : 19~ 22
Bt 棉棉子中 Bt 蛋白定量检测方法建立及变化规律研究
徐宝梁1, 2 , 苏 宁2 , 袁 飞1 , 王因霞1 , 陈 颖2 , 吴亚君2 , 张青文1* ( 1. 中国农业大学, 北京 100061; 2. 中国检验检疫科学研究院, 北京 100025)
Enviro logix kit w it h Bt prot ein reference st andards could be used t o det er mine t he Bt prot ein quant itat ively. Agdia kit w it h high sensit ivit y w as able t o det ect t he Bt pr ot ein in cot t onseed, kernels and shells qualit at iv ely . T he quant it at ive met ho d w ith high sensit ivity fo r t he det er minat ion of Bt prot ein w as est ablished in this st udy based on t he char act erist ics of Env irolog ix kit and Ag dia kit , and the
的 OD 630与其 Bt 蛋白含量间有很好的相关性, 相关系数大于 0. 99。据此, 利用 A g dia 试剂盒
测得 99B、33B 棉子、棉子仁、棉子壳在 12 个月的贮存过程中 Bt 蛋白含量的变化规律, 棉子、
棉子仁中 Bt 蛋白含量降低一半, 棉子壳中 Bt 蛋白含量降低四分之一。
关键词: Bt 棉棉子; Bt 蛋白含量; 变化规律
收稿日期: 2005- 03- 02
作者简介: 徐宝梁( 1963- ) , 男, 硕士, 高级工程师, x ubao l@ y aho o. co m. cn; * 通讯作者
基金项目: 科技部社会公益
棉 花学报
18 卷
degradat ion rules of Bt prot ein in 99B and 33B cot t onseeds, kernels and shells in the pro cess of st orage w ere described in Bt amounts. T he cont ent s o f Bt pro t ein in cot t onseed shells decreased rapidly in the pro cess of st orag e, and the co nt ent s of Bt pr ot ein in cot t onseeds and kernels decr eased more rapidly in the st orage. When cot to nseeds, kernels and shells w ere used as mat erials in the st udy relat ed t o the Bt prot ein, t he degradat ion of Bt prot ein in t he st or ag e pro cess of those mat erials should be taken int o acco unt. When t he Bt prot ein cont ent s in cot t onseeds and kernels were det erm ined using Enviro logix kit and Agdia kit , t he Bt prot ein cont ent s w ere not linearly co rrelat ed w it h net opt ical density at low diluted t im es, but w ere linearly corr elat ed w it h t he net optical density at high dilut ed t imes. As such, fo r t he det er minat ion of Bt pro tein t he ex tr act io ns of cot t onseeds and kernels sho uld be dilut ed unt il the Bt prot ein concent rations w ere linearly correlat ed w it h t he net o pt ical density m easured using the kit s. T he ex t ract ion sho uld be dilut ed about 100 t im es, and thus t he Bt prot ein cont ent s measur ed using t he kit s w ere real, o therw ise t he cont ent o f Bt prot ein in co tt onseeds and kernels determ ined using t he kit s w ould be low er t han that norm al.