Cloning,Expression Analysis And Vector Construction Of The Plasma Membrane Intrinsic Protein Genes In Lotus Root

Lotus root(Nelumbo nucifera Gaertn),origining from China and India belongs to family Nymphaeaceae,and it is used as an aquatic herb vegetable.Lotus is grown in Southeast Asia,Japan and China.Now lotus root has been widely cultivated in China for multiple purposes.The products of lotus root,lotus seeds and lotus root starch are very popular in the daily diet because of its richness in nutrients.Currently,nomal cultivars of lotus root used in product are sensitive to salt,while salt-tolerant resources exist in wild cultivars.With the expansion of soil salinization and the area along the beach in China,planting of salt-tolerant varieties of lotus root in salinized soils will not only help to improve efficiency of land usage,but also improve local environment.Plasma membrane instrins protein(PIP)is one of the most studied aquaporins,and it was shown that PIP plays an important role in enhancement of plant salt tolerance.Due to the limitations of cell membranes,PIP increases plant salt tolerance by mediating the transcellular transport of water in leaves and roots.Therefore,in this study,genes of the PIP family were cloned from salt-tolerant wild lotus and their expression characteristics derived from various stresses were studied.At the same time,the expressing vectors of three salt-induced genes(NnPIP1-2,NnPIP2-1 and NnPIP2-7)were also constructed.We obtained following results:1.Eight PIP genes were cloned from salt-tolerant wild lotus root(H5)using RT-PCR technology.The full length of these lotus PIP genes was between 843 bp and 864 bp.By comparing against to the NCBI database,we found that PIP family genes obtained in this study showed high similarity with that of Arabidopsis,maize and rice,and the similarity between genes of lotus PIPs was from 67%and 90%.Therefore,the genes of lotus were designated as NnPIPs.In addition,NnPIPl and AtPIPl,NnPIP2 and AtPIP2 had great homolog than that of NnPIPl and NnPIP2,so we believed that NnPIPl and NnPIP2 belonged to two subgroups of the same gene family.At the same time,the characteristic of gene nucleiotide was also analyzed,it was found that each member of NnPIPs had 6 alpha-transmembrane helices and 5 linked loops(A-ring-E loop),and there was an NPA(Asn-Pro-Ala)domain on both B and E loops.These features of gene were critical for water and some small molecules,such as CO2 transport.2.Used ?-actin as an internal standard,semi-qRT-PCR and qRT-PCR were carried out to study the expression profiling of NnPIPs in H5 treated with NaCl,mannitol,ABA,low temperature and PEG for 24 h.For NaCl treatment,the expression of NnPIP1-2 was up-regulated at 0-24 h after treatment and the transcriptional level reached the maxium at 24 h.The expression of NnPIP2-1 and NnPIP2-7 was also increased at 0-12 h,while NnPIPl-3 and NnPIP1-4 decreased expression.NnPIP1-1 and NnPIP2-8 were not induced by NaCl because the expression had no significant change during the whole treated period;For 200 mM mannitol treatment,the expression of NnPIP2-8 was significantly increased after 12 h treatment,and reached the maximum at 18 h.Others genes including NnPIP1-1,NnPIP1-2,NnPIP1-3,NnPIP1-4,NnPIP2-1 and NnPIP2-7 were not significantly changed in mRNA level.For treatment of 50 ?M ABA,the expression of NnPIP1-3,NnPIP2-1,NnPIP2-7 and NnPIP2-8 was enhanced after treatment of 6 h to 18 h respectively,and NnPIP1-1,NnPIP1-2 and NnPIPl-4 were not significantly changed.For low temperature(4?)treatment,NnPIP1-1 and NnPIP2-7 was increased in expression and reached the maximum at 18 h.The expression of NnPIP1-2,NnPIP1-3 and NnPIP2-8 showed no significant change within 24 h after treatment,the expression of NnPIP1-4 and NnPIP2-1 was decreased within 0-24 h;Treated with 10%PEG 6000 for 24 h,we found that the expression of NnPIP1-1,NnPIP1-2,NnPIP1-3,NnPIP1-4,NnPIP2-1,NnPIP2-7 and NnPIP2-8 were enhanced within 24 h repectively,among which,NnPIP1-4 was firstly increased and then decreased in expression.Generally,the above data showed that NnPIPs were responsive to various stesses condtions.3.In this experiment,over-expressing vectors of three salt-induced genes of PIP family(NnPIP1-2,NnPIP2-1 and NnPIP2-7)were constructed with pAMBIA1301 to further study gene function.After identification with digestion with double enzymes and PCR results,we believe that the overexpressing vectors of pAMBIA1301-NnPIP1-2,pAMBIA1301-NnPIP2-1 and pAMBIA1301-NnPIP2-7 were successfully constructed.