摘要
目的通过研究转rolC基因八棱海棠不同株系的转基因拷贝数,在转录水平上的表达丰度,以及转基因组培苗的生物学特性,为进一步深入阐明rolC基因在八棱海棠中的表达机制和培育优良的苹果砧木奠定理论基础。方法以通过gus染色和PCR检测的3个转rolC基因八棱海棠株系组培苗为试材。Southern杂交鉴定rolC基因整合拷贝数。Northern杂交鉴定rolC基因在转录水平上的表达丰度。调查转基因植株组培苗在含有不同种类和浓度的植物生长调节剂的培养基上茎段增殖、叶片再生、生根能力等生物学特性;将生根后的组培苗进行炼苗,移入温室4个月后,研究株高、节间数、节间长度、叶面积等生物学特性。结果Southern杂交结果表明,rolC基因分别整合进入3个八棱海棠株系基因组,其中株系20a和33a分别获得了1个rolC基因拷贝,株系20b获得了2个rolC基因拷贝。Northern杂交结果表明,3个转基因株系中的rolC基因均在转录水平上得到了表达,且该基因在单拷贝株系的表达丰度高于双拷贝株系。转基因组培苗生物学特性研究结果表明:(1)3个转rolC基因八棱海棠株系茎段增殖系数、叶片再生率、生根所需外源激素浓度均显著低于对照植株,单拷贝株系的以上指标亦显著低于双拷贝株系。(2)3个转基因八棱海棠株系组培苗平均生根数显著高于转基因株系,双拷贝株系显著低于单拷贝株系;根长情况正好相反;对照植株根粗和转基因株系根粗之间均无显著差异。(3)3个转基因八棱海棠株系的株高、节间长度、节间数、叶面积均显著小于对照植株。其中单拷贝株系显著小于双拷贝株系。结论rolC基因整合进入3个八棱海棠转基因株系基因组,并分别在转录水平上得到表达。外源rolC基因的表达导致转基因植株体内内源激素含量和植株形态学的改变,且rolC基因的整合拷贝数对它的表达有一定的影响。
[ Objective ] The purpose of this research was to investigate the copy numbers, expression quantities at transcription level and biology ofrolC gene in transgenic Malus robusta Rehd. The study also aimed to provide basic information for investigating the expression ofrolC gene in genome ofMalus robusta Rehd and breeding excellent apple rootstocks. [Method] Three clones of rolC-transgenic Malus robusta Rehd., which have been firstly screened through the gus and PCR tests, were selected for further investigation. Molecular hybridizations were made to identify rolC gene integrated copy numbers and expression quantities at the transcription level in Malus robusta Rehd. The biology of transgenic Malus robusta Rehd, such as shoots proliferation, leaf differentiation and rooting abilities, were surveyed. Other characteristics of transgenic Malus robusta Rehd, such as plantlet height, nodes number, the length of internodes and leaf area, were researched after they grew in a greenhouse for 4 months. [ Result ] Southern hybridization demonstrated that rolC gene had been integrated in the genome of three clones of rolC-transgenic Malus robusta Rehd. Two of these transformants had a single copy and one had double copies of the integrated rolC gene. Northern hybridization showed that the expressions of rolC gene in transgenic clones were influenced via different integrated copy numbers. At the transcription level, its expression quantity in the clone with double rolC copies was less than that of other two clones with a single rolC copy number. Studies revealed that： （1） The rates of shoots propagation, rooting and leaves regeneration of all three rolC-transgenic Malus robusta Rehd. clones were lower than that of the non-transgenic plantlet at significant level. And these indexes of clones with one rolC gene copy were significantly lower than that of the clone with two rolC gene copies. （2） The root numbers of these three rolC-transgenic Malus robusta Rehd clones were more than that of the non-transgenic plantlet at significant level. Among transformants, the data showed that clones with one rolC gene copy were significant larger than that of the clone with two rolC gene copies. The root length of the non-transgenic is significant longer than that of three transformants. This length of one rolC gene copy clones is significant shorter than that of two rolC gene copies clone. The root diameter had no difference between the non-transgenic plantlet and the transgenic clones. （3） All three rolC-transgenic Malus robusta Rehd clones display the obvious characteristic of roIC phenotype, such as dwarf shoot, shorter internodes, smaller leave area, plantlet shorter than non-transgenic clones. And the clones carrying one rolC gene copy were significant shorter than the clone with two rolC gene copies. [ Conclusion] The rolC gene has been integrated into the genome of Malus robusta Rehd. It was expressed at the level of transcription. The expression of exogenous rolC gene changes endogenous hormone contents and morphology of transgenic Malus robusta Rehd. The integrated copy number ofrolC gene played an important role at the expression ofrolC gene in transgenic clones.
出处
《中国农业科学》
CAS
CSCD
北大核心
2006年第12期2563-2569,共7页
Scientia Agricultura Sinica
基金
国家自然科学基金资助项目(30370987)
江苏省科技研究资助项目(BJ2003309)
