关键加工因子（温度、水分和压力）影响饲料转基因成分的解构规律研究

Due to clarify a series of questions pertaining to the safety of transgenic feed, it has become urgently necessary to trace the entire process from production to consumption of transgenic feed, as well as to study and discuss the changes sustained by genome DNA and transferred exogenous genes in these processed feed products. Hitherto, numerous researches on transgenic feed are mainly con?ned within ?elds of safety evaluation and detection of transgenic components in transgenic raw materials and processed products, especially, establishment and improvement of various detecting methods, however, few work has been done on DNA degradation, especially transferred exogenous genes in feed products during the processing of transgenic crops. In this study, changes in the DNA length of transgenic components in GM crops (soybean, maize, cotton and rapeseed meal) in each key factor- temperature,water content and pressure during the feed processing were investigated in order to understand the effect of different processing factors on genomic, and exogenous DNA, and to explore the degradation mechanism of endogenous gene and exogenous gene. It is our purpose to provide more knowledge for improvement of detecting method used for the identi?cation of transgenic feed products, and to provide a scienti?c data for transgenic identi?cation and security evaluation in our country and to make identi?cation systems and security evaluations more rational and scienti?c. At the same time, we hope to enrich the theoretical studies of these new feed resources- transgenic feed- and to provide guidance in controlling their processing.

为澄清转基因饲料安全问题的真相，在对"从生产到消费"的全过程质量管理与溯源时迫切需要探讨转基因饲料在加工过程中DNA和内外源基因降解变化规律。目前，饲料加工因子（温度、水分和压力）对转基因饲料DNA和内外源基因降解变化规律影响及其导向的转基因成分检测特殊性规律影响的研究很少。本项目采用改良SDS、CTAB和试剂盒法等多种DNA抽提方法，建立适合饲料深加工产品的最佳DNA提取方法，以此为基础探讨分析饲料加工因子（温度、水分和压力）对核酸DNA降解变化规律；通过LAMP/LAMP-LFD、HRCA、多重PCR、基因芯片方法分析探讨饲料加工因子（温度、水分和压力）对内外源基因降解的影响及变化规律及其导向的饲料转基因成分检测的特殊性规律。本研究为饲料转基因成分检测技术的改进提供技术支撑和科学依据，也对转基因饲料的加工生产和安全评价具有指导意义。

按照合同任务书要求，完成全部任务。主要研究成果如下：.（1）饲料加工因子对转基因棉粕的影响及其降解规律的研究.研究了温度、水分和压力对转基因棉粕核酸降解规律的影响，结果表明：随着温度升高，转基因大片段逐渐降解，但降解幅度不同。同等条件下，湿热处理对降解作用大于干热处理。105℃不同时间干热及湿热处理后，各片段降解差异不大。在湿热膨化中，温度越高，大片段降解越明显。不同水分比例对各片段的降解差异不大。干热处理后外源/内源基因拷贝数比没有明显的变化规律。105℃湿热处理不同时间后，外源/内源基因拷贝数比没有明显的变化规律。温度升高时，外源/内源基因拷贝数比逐渐降低。不同比例水分湿热膨化后，外源/内源基因拷贝数比没有明显的变化规律。膨化温度升高后，外源/内源基因拷贝数比逐渐降低。.（2）饲料中高破坏性、重解构转基因成分检测的特殊性规律研究.研究了温度、水分和压力对转基因豆粕核酸和蛋白质降解规律的影响，结果表明：不同加工方式会对豆粕内、外源基因降解的影响不同，高温高压高湿对小片段Lectin（118 bp）和CP4-EPSPS（155 bp）影响相对较小；414 bp Lectin基因在干热（75~90℃）处理30 min、湿热和膨化（165℃ 39%）处理后，彻底降解；483 bp CP4-EPSPS基因经干热、湿热、膨化（120℃，39%）后未检测到。Lectin和CP4-EPSPS在温度90~165℃的降解率分别为0.4%~92.1%、6.1%~84.0%；高含水率会在一定程度上缓解内、外源基因的降解。iELISA和Western结果表明膨化豆粕中CP4-EPSPS蛋白含量随着温度和含水率的升高而降低，与DNA降解规律一致。.根据内外源基因降解变化规律，建立了饲料深加工产品中痕量、高通量转基因分子特征精准检测技术mHRCA-RDB与mPCR-RDB。 .（3）适合饲料深加工产品高效快速DNA抽提技术的研究.建立了改良CTAB法、试剂盒法和SDS法，结果表明：SDS法提取效果最好，CTAB法次之，试剂盒方法提取效果最差。SDS法适用于饲料原料及其深加工或成分复杂的产品。.合同书之外，构建了含有10个目标基因的质粒标准分子，覆盖了在饲料原料（豆粕、玉米和棉粕）中10个具有筛选、品系、基因特异性的目标基因，代替标准物质用于转基因生物饲料深加工产品定性、定量检测的阳性对照。