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采用DDRT-PCR技术,以棉花盛花期项尖叶片cDNA为材料,对上海生物工程公司合成的专用于基因差异显示分析的3个锚定引物和全套26个随机引物进行筛选,最后选择了15个扩增差异带丰富的随机引物。采用3个锚定引物和这15个随机引物组成的45对引物组合对24个抗虫棉杂交组合及其10亲本盛花期叶片cDNA进行扩增和差显, 2次扩增重复率达70．1％,表明在扩增过程中存在较高的假阳性,通过重复PCR扩增,统计稳定扩增的条带,可减少假阳性干扰。根据基因表达方式,将其划分5种模式:M1为双亲表达沉默,双亲出现条带而杂种没有条带; M2为单亲表达沉默,带仅出现在亲本之一,包括仅母本有带而父本和杂种无带和仅父本有带而母本和杂种无带2种表达方式;M3为杂种特异表达,带仅出现在杂种,双亲无带;M4为单亲表达一致,带在双亲之一和杂种中出现,而在另一亲本中不出现,包括母本、杂种中有带而父本无带和父本、杂种中有带而母本无带2种方式;M5为基因表达一致,带在双亲和杂种中均出现。差显表达模式比例与产量性状和杂种优势分析表明:M4与所有产量性状均呈正相关,并且与单位面积铃数相关达显著水平,其他各种模式与杂种产量性状表型值均未达到显著水平;M2与单位面积铃数杂种优势呈显著负相关,M3与皮棉产量杂种优势早显著正相关。上述结果表明,盛花期叶片中的基因显性表达和杂种特异表达有利于产量形成和杂种优势发挥。
Using DDRT-PCR technique, we selected three anchor primers and a set of 26 random primers which were synthesized by Shanghai Bioengineering Co., Differences with amplification of random primers. 45 pairs of primer combinations consisting of three anchored primers and 15 random primers were used to amplify and differentiate the 24 hybrid cotton combinations and their 10-parent full-flowering leaf cDNA. 1%, indicating that in the amplification process there is a high false positive, amplified by repeated PCR amplification, statistical amplification of the band, can reduce the false positive interference. According to the way of gene expression, it is divided into five kinds of patterns: M1 is silenced by parents, parents have bands but hybrid does not have bands; M2 is silenced by single parents and only occurs in one of the parents, The two hybrids were banding with no banding and only male with banding and no banding with mother and hybrids. M3 was hybrid-specific with banding only in the hybrid with no banding in the parents. M4 was single-parentally expressed with one of the parents And hybrids, but not in the other parents, including maternal, hybrid and male parental bandage without paternity, hybrids and maternal band without banding two ways; M5 gene expression is consistent, Bands appear in both parents and hybrids. The ratio of the differential expression pattern and yield traits and heterosis analysis showed that: M4 was positively correlated with all the yield traits, and significant correlation with the number of bolls per unit area, and the other types of phenotypic traits and hybrid yield traits did not reach significant levels ; M2 had a significant negative correlation with the heterosis of boll number per unit area, and had a significant positive correlation with the heterosis of lint yield. The above results show that the dominant gene expression and hybrid-specific expression in the full-flowering leaves are conducive to yield formation and heterosis play.