非组培依赖的发根农杆菌介导的薄壳山核桃转化体系构建

Construction of Agrobacterium rhizogenes-mediated transformation system of Carya illinoinensis without dependence on tissue culture

【目的】建立一种简单高效的农杆菌介导薄壳山核桃Carya illinoinensis的转化体系。【方法】以薄壳山核桃钟山的幼苗为材料,采用四因素(菌种、菌液浓度、处理部位、苗龄)三水平正交试验进行农杆菌侵染薄壳山核桃茎部诱导发根,并通过DNA检测及GFP荧光验证。【结果】影响发根农杆菌侵染薄壳山核桃生根诱导率的因素大小为苗龄>处理部位>菌液浓度>菌种,OD_(600)为0.8的K599发根农杆菌于材料子叶期时侵染距离种子3~5 cm处,毛状根植株诱导率达56.5%,阳性毛状根植株诱导率为45.2%。【结论】建立并优化了非组培依赖的发根农杆菌介导的薄壳山核桃转化体系,为薄壳山核桃新品种培育、利用基因工程改良农艺性状奠定基础。

【Objective】Carya illinoinensis,also known as pecan,is originated from the North America.Pecan is one of the most economically valuable woody species and a high value tree for edible nut production in the world.Current pecan transformation methods are time-consuming and low efficiency.Due to the lack of simple and efficient genetic transformation system for pecan,it is urgent to develop a simple and highly efficient transformation system so that the function of genes could be studied through the transgenic system quickly and efficiently.In recent years,an Agrobacterium rhizogenes mediated transformation method has been developed,which is less time-consuming and species dependent in generating transgenic plant tissue.To obtain a simple and efficient A.rhizogenes mediated transgenic system,we established a transgenic root system with pecan seedlings.【Methods】Taking the seedlings of Carya illinoinensis(Wangenh.)K.Koch‘Zhongshan’in a forest nursery as materials,the effects of strain,the concentration of bacterial liquid,treatment site,and seedling age were studied on the infection of pecan.The GFP expression vector pCAMBIA-35S:GFP was used to test the transgenic-positive roots.The plasmid pCAMBIA1300-35S:GFP was transformed into A.rhizogenes K599,A.rhizogenes C58C1,or A.rhizogenes MSU440,respectively.Then,a design of experiments(L9)with four factors and three levels was carried out.The factors studied were:strain type(ST),bacterial liquid concentration(BC),treatment site(TS),and seedling stage(SS).A genetic transformation system for pecan was performed and,in each of them,strain type(STK599,STC58C1 and STMSU440),bacterial liquid concentration(BC0.4,BC0.8 and BC1.2),treatment site(TS0-1,TS1-3 and TS3-5),and seedling stage(SS0,SS2 and SS4)were measured.The seedlings were grown in an artificial climate chamber(luminance 200 lx,photoperiod 12 h light,and humidity 70%±5%).Seeds were collected and sown immediately on a sowing substrate for germination.After germination,the germinated seeds were sown in plastic pots containing cropping substrate.The vegetative stage(2-3 months after seed sowing)was selected as a transgenic receptor.All seedlings were generated via injection or a blade.The seedlings were covered for a dark treatment(48 h dark)after infestation.For efficiency analysis,hairy rooting efficiency,hairy rooting number,and transformation rate were analyzed.Hairy rooting efficiency was estimated as rooting rate per explant.The hairy rooting number was counted as the root number per explant.Transformation rate was calculated as positively transformed rooting per explant.All statistics were recorded at 30 days post-infestation.For detection of GFP fluorescence,roots were observed with a fluorescent microscope or a handheld fluorometer.The inserted GFP sequence was verified by PCR.After confirmation,the untransformed hairy roots and primary roots were cut manually.Transgenic hairy roots were maintained for seedling growth.【Results】For both infestation methods,the final transgenic rate was not different between needle injection and blade scratch.For the convenience of operation,we choose a blade to scratch in the following experiments.The callus tissues formed on the infestation sites after 10-15 days post-infestation using A.rhizogenes.The hairy roots developed from the callus after 10-15 days.The GFP fluorescence was observed both in the callus and hairy roots.According to the results of orthogonal experiments,the factor order of influencing the transformation efficiency was the seedling stage,treatment site,bacterial liquid concentration,and strain type.The optimal influential variables,in this case,were strain type for STK599,bacterial liquid concentration for BC0.8,treatment site for TS3-5,and seedling stage for SS0.The results showed that hairy rooting efficiency was 56.5%,and transgenic rooting rate was 78.5%,while the final transformation rate was 45.2%.The GFP fluorescence was detected in the whole roots of transgenic pecan plants.The inserted GFP fragment was also confirmed by PCR amplification of the GFP coding sequence.We found that it was vital to bury the infestation sites in the substate during the whole experiment.If the infestation sites were exposed to air,the hairy roots could not emerge from the cut surface of the shoots.The transgenic-positive pecan hairy roots were able to develop and become thicker and more lignified.Meanwhile,the original taproot growth of pecan was arrested.The seedlings grew normally without the transgenic-negative roots and taproots.【Conclusion】We successfully established an efficient and simple A.rhizogenes mediated transgenic root system for pecan,which was not dependent on the tissue culture.It was proved that high efficiency transform could be achieved if an appropriate seedling stage was chosen for the specific infestation site for Agrobacterium-mediated transformation.Our study provides useful information for the construction of a simple and high efficient transformation system for hairy root transgenic crop production.This transgenic system is a powerful tool for gene functional characterization.This study also provides a foundation for cultivating new varieties of pecan and the improvement of agronomic characteristics by genetic engineering.