Preliminary Study On CmPIP Associated With Stress Related Of Chrysanthemum Morifolium

Chrysanthemum morifolium is perennial root herb,a number of the composite family.Ground-grow chrysanthemum is a new cuitivar of ground-cover chrysanthemum,which has characteristics of short plants,extensive management,high ornamental value,strong stress resistance.Plasma membrane intrinsic proteins as a number of aquaporin super family MIP family,play a important role in plant stress resistance.According to the known study of PIPs aquaporin related stress resistance,using the bioinformatics method to analysis known sequences of PIP gene in other species,we cloned the sequence of CmPIP genes.Then we processed functional analysis of CmPIP genes,expect to find out expression pattern of CmPIP genes under stress condition.We acquire the transgentic regeneration Ground-grow chrysanthemum plant,which have better resistance of stress.Our purpose is to make a better understanding of plant stress resistance mechanism,to cultivate stress resistance Chrysanthemum morifolium.The overall results are showed as follows:1.Experimental plants come from Institute of flower;Northeast Forestry University.Two full length cDNAs named CmPIP1 and CmPIP2 was cloned from Chrysanthemum morifolium by RACE(rapid amplification of cDNA ends)technology.Full length of CmPIP1 gene is 1170bp,contains a 864bp open reading fram,coding 287 amio acid.Full length of CmPIP2 gene is 918bp,contains a 858bp open reading fram,coding 285 amio acid.Using NCBI blast,CmPIP1 showed similarity to gerbera hybrid by 99%,and CmPIP2 showed similarity to vitis vinifera by 95%.Homology analysis showed that CmPIP1 and CmPIP2 shared high homology with maize and poplar respectively.Bioinformatics analysis showed CmPIP 1 and CmPIP2 both have MIP structural domain,and NPA motif.That confirm CmPIP1 and CmPIP2 are aquaporin.2.Onion epidermal cell subcellular localization asssy showed CmPIP1 and CmPIP2 proteins are localized in the plasma membrane.3.According to the CmPIP1 and CmPIP2 gene sequences,we constructed pH7WG2D-CmPIP overexpression vector and pH7GWIWG2D-CmPIP RNAi interference vector.Then transferred into Agrobacterium tumefaciems LBA4404 by tri-parent conjugation.Agrobacterium tumefaciems used for plant transformation.4.Yeast two hybrid analysis confirmed that CmPIP1 has no interaction with CmPIP2.5.For CmPIP gene analysis,WT Chrysanthemum morifolium were grown in 200mM/L NaCl Hoagland,s solution for up to 72h.We collected leaves and roots at 0,4,8,12,24,72h for expression analysis.Real-time fluorescence quantification results showed that CmPIP1 gene was downregulated by salt stress in leaves.While,in leaves,the transcripts of CmPIP2 increased gradually from 0 to 4h treatment,then decreased sharply after 4h treatment.In the early 48h,the CmPIP2 was upregulated in roots,reached the peak value at 8h.6.We transferred the pCAMBIA1301-PMI-LcPIP overexpression vector into Ground-grow chrysanthemum ’huoyan’ from Northeast Forestry University by agrobacterium-mediated method.Then confirmed positive plants by PCR and GUS dyeing.We acquire the same stripe with positive control.GUS dyeing showed that pCAMBIA1301-PMI-CmPIP overexpression vector has expressed in transgentic plants.7.Quantitative Real-Time PCR analysis showed that under salt stress,in transgenic plants,CmPIP1 and CmPIP2 have better salt response ability than WT plants.Exogenous LcPIP gene can upregulate endogenous CmPIP genes.8.We test the activity of POD and SOD,and capatity of MDA in both transgenic and wild type Chrysanthemum morifolium to identify the transgenic Chrysanthemum morifolium ability of salt stress tolerance.We found transgenic Chrysanthemum morifolium has better salt stress tolerance than wild type Chrysanthemum morifolium.