Functional Analysis Of The Alternative Splicing Isoforms Of The NBS1 Gene In Arabidopsis

DNA damage is an essential factor affecting plant growth and development,and there are two main repair pathways: homologous recombination and non-homologous end joining.NBS1(Nijmegen breakage syndrome 1)is a key regulator involved in both repair pathways,interacting with MRE11(Meiotic recombination 11)and RAD50 to form the MRN complex,which is important for maintaining cellular genome integrity.In our laboratory,we found that the NBS1 gene in Arabidopsis has two types of variable splicing,one is the normally spliced NBS1 gene,and the other is the form with variable splicing(AS)at the 3’ end.The main results of this study,which focused on the functional differences between Arabidopsis NBS1 and its variable splicing form NBS1-3 in DNA damage repair,are as follows:1.The effects of DNA damage treatment of wild-type(WT)Arabidopsis seedlings by chemical DNA damage agent MMS(Methyl Methanesulfonate)and physical UVB(Ultraviolet radiation b)on their growth and development and the expression of the three MRN complex components were investigated.In order to provide a more visual demonstration of the extent of DNA damage,a PI staining solution was also used to observe the staining of root tip cells treated with MMS.The results showed that the DNA damage treatments used in this study were effective and all resulted in DNA damage in Arabidopsis seedlings,leading to retarded growth and triggering their DNA damage repair response.The gene expression of MRN complex components increased and then decreased with increasing MMS treatment concentration and UVB irradiation time,and after MMS treatment,PI staining of Arabidopsis seedlings root tips revealed a lamellar distribution of red areas,dead cells with DNA damage,which was also an indication that the cells in the root tips of Arabidopsis seedlings had DNA damage.These results suggest that DNA damage has occurred in Arabidopsis seedlings after treatment with MMS or UVB,and that the damaged DNA is repaired by either homologous recombination or non-homologous end joining pathways involving MRN complex.2.NBS1-3 is missing 27 bp between the 1000 and 1028 bp than NBS1,because NBS1-3 has an AS at the 3’ end and truncates the 9th intron together with the 10 th exon junction.The transcript lacking 27 bp was produced by the AS at the 3’ end of NBS1-3.Preliminary analysis of the functional differences between the two using subcellular localization showed that both NBS1 and NBS1-3 proteins localize to the nucleus,indicating that the missing 9 amino acids do not affect the expression position of the NBS1 protein in plants,nor the MRN complex components MRE11 and RAD50 proteins in plants.This may be because amino acids 2 to 124 are the putative nuclear localization sequence(NLS)of the NBS1 protein,whereas NBS1-3 lacks 9 amino acids between amino acids 333-343,so the absence of these 9 amino acids does not influence the subcellular localization of NBS1.3.Ten NBS1 and seven NBS1-3 overexpressing transgenic Arabidopsis strains were obtained by the flower dip method,and the Arabidopsis NBS1 mutant nbs1 was identified as a pure mutant by the triple primer method.Two strains with high NBS1 expression were selected from each of the NBS1 and NBS1-3 overexpression transgenic Arabidopsis strains and treated with 0.6 m M MMS for DNA damage,and the extent of DNA damage in the root tips of Arabidopsis seedlings was observed by PI staining.The results showed that the NBS1 mutant nbs1 had the largest relative damage area and NBS1-3-OE-1 had the smallest relative damage area,which was significantly smaller than that of WT,suggesting that the NBS1 gene plays an important role in the repair of DNA damage caused by MMS and that NBS1-3 may play a greater role than the NBS1 gene in this process,that is,the missing 9 amino acids may enhance the role of NBS1 protein in DNA damage repair.In this study,we investigated the effect of the missing 9 amino acids on the function of the NBS1 protein by genetic and molecular biological methods,starting from the investigation of the functional differences between NBS1 and its alternative splicing isoform NBS1-3.An effective DNA damage treatment and a method to detect the extent of DNA damage were first established,then the sequences of both were analyzed and subcellular localization was observed,but the missing 9 amino acids did not affect the expression localization of their proteins.The NBS1 and NBS1-3 overexpression vectors were transferred into WT Arabidopsis to obtain transgenic Arabidopsis material,and the Arabidopsis NBS1 mutant nbs1 was obtained from the Arabidopsis mutant repository Ara Share.This Arabidopsis genetic material was treated with the previously established DNA damage treatment method,and the staining of Arabidopsis seedling root tips was observed by PI staining.We first demonstrated the important role of the NBS1 gene in DNA damage repair in Arabidopsis seedlings and speculated that NBS1-3 may play a greater role than NBS1,providing a theoretical basis for studying the DNA damage repair mechanism.