The Functional Analysis Of Nicotiana Sylvestris CBL Family Gene NsylCBL4 And NsylCBL6 Involved In Abiotic Stress

Calcium(Ca²⁺)is the second messenger in plants,which is involved in the regulation of plant growth and development,as well as the biological and abiotic stress response.However,calcineurin B-like protein?CBL?is considered to be an important Ca²⁺sensor in plant-specific calcium signalling pathways.So far,our group has identified 12 CBL proteins from Nicotiana sylvestris.In this study,NsylCBL4,NsylCBL6,NsylCBL7,and NsylCBL8 gene overexpression transgenic materials were constructed and the functions of NsylCBL4 and NsylCBL6 genes were further explored.?1?Sequence analysis of four NsylCBLs proteins:The CDS sequence of NsylCBL4 encodes 234 amino acids,among which there are three Ca²⁺binding motifs in the amino acid sequence.The CDs sequence of NsylCBL6 encodes 211 amino acids,among which there are three EF-hand structures.Besides,NsylCBL6 has a high similarity with AtCBL7in Arabidopsis,about 71.35%,and 72.51%with Pe CBL3 in poplar.?2?Four gene overexpression materials were obtainedThe hypothetical 35S:NsylCBL4 tobacco transgenic plants were screened by the presence of NsylCBL4,and then the actin was used as the reference gene to further screen the low expression and high expression of the target gene by genome and semi-quantitative PCR analysis.Among all the 24lines of 35S:NsylCBL4,OE lines,15,and 24 were selected for functional analysis.Firstly,abiotic stress analysis was conducted to evaluate the stress tolerance of T2 35S:NsylCBL4 transgenic tobacco.Similarly,The NsylCBL6 gene was transferred into Arabidopsis thaliana by floral depth method.T1generation was detected by genomic PCR and further confirmed by semi-quantitative and fluorescent quantitative PCR.In addition,resistance screening methods were used to further select homozygous T2and T3 lines,and T3 seeds were collected for functional analysis of NsylCBL6.?3?Effects of NsylCBL4 on leaf development and response to osmotic stress:The overexpression of NsylCBL4 changed the leaf morphology of transgenic tobacco,which was characterized by the decrease of plant height and leaf stenosis.Moreover,it makes the epidermal cell layer concentrate,which increases the chlorophyll content of leaves and affects the photosynthetic rate.Daily observation showed that the leaf morphology of the transgenic lines atf 400 mM and 500 mM sorbitol media was significantly different,and there was a significant change on the leaf disc.The transgenic lines were more sensitive to osmotic stress,and the leaf disc was completely blocked.?4?NsylCBL6 and Nitrate?NO₃^-?signal transduction in plant roots:Under low NO₃^-stress,the overexpression of NsylCBL6 gene increased the number of lateral roots,and the elongation of roots in transgenic Arabidopsis.Therefore,NsylCBL6 may be involved in the nitrate signalling pathway in Arabidopsis root.In conclusion,it is suggested that NsylCBL4 is an important gene that affects leaf growth and development and response to osmotic stress,and NsylCBL6 is a key gene involved in nitrate signal transduction.The above work provides more data support for the study of the CBLs gene family in plant growth and development and abiotic stress response.