Sabina Vulgaris Germplasms Growth And Drought Tolerance Evaluation As Well As Function Study Of Its Zn²⁺ Binding Protein SvCML1

Having genes with clear physiological function is the basis of crop molecular breeding.The abundant wild plant resources are natural genetic library.The current high throughput sequencing technology makes it possible to explore wile plants'genetic resources.Sabina vulgaris is an creeping shrub in the Cupressaceae,distributed in the desert region of the northern China.It shows strong tolerance to drought and nutrient-deficient soils and is widely used in ecological greening projects in the northern China.This study was based on collection of wild Sabina vulgaris germplasm in the northern China.The growth and drought response of those germplasms were evaluated.Initially we expected to find Sabina vulgaris specific Ca²⁺binding protein-encoding genes with transcriptome.Unexpectedly,we found that SvCML1?Calmodulin Like1?predicated having Ca²⁺binding motif,actually was an protein specifically binding to Zn²⁺.We further explored the physiological role of Sv CML1 in the Zn²⁺-dependent growth.The main contents and conclusion of this study are followed:?1?Totally 221 Sabina germplasms were collected from four natural conservation regions in Inner Mongoila and three regions in the GanSu,QingHai,ShanXi,which were propagated in the normalized growth condition in the Hohhot Helin experimental base.After 3.5-year,the branches length of 49 germplasms were measured.Average and standard error were calculated from at least 10 replicates for each germplasms.It was found that lines from ShanXi HengShan and Inner Mongolia TaoLi showed largest growth with 1.5 to 2 meters of branches;the slowest are the lines from Inner Mongolia HeLanShan and Gan Su-TianZhu QiLianShan with 0.5 to 1meter of branches.?2?Forty-nine germplasms were treated with 6 and 11-month drought.Afterward,the plants were re-given water and materials were collected as control after 1-month.For each of germplasms,at least three growth consistent branches from the above-ground of grafted plants were collected for drought resistant index measurement.The contents of proline,MDA and activities of POD and SOD were measured with materials from the branches of these germplasms.Our basic standard for evaluating drought tolerance is that:with drought treatment,the higher of proline content and activities of POD and SOD,as well as the lower of the MDA content,the stronger of the germplasm's drought tolerance.In contrast,the smaller of the increasing extents of the proline contents and POD,SOD activities,as well as the larger of the increasing extent of the MDA content,the weaker of the germplasm's drought tolerance.With this standard,it was hard to determine the drought tolerance ability of most of the tested germplasms,since there were no clear patterns of the four index changes.?3?Germplasms with clear drought-tolerance characteristics are the#7 from Inner Mongolia TuKe:under the drought treatment,its MDA content had no significant rise,but the proline content and SOD activity increased significantly.In addition,the#30 from Inner Mongolia TongDeGeEr and#8 from Inner Mongolia TaoLi and#5 from ShanXi HengShan,had no change of MDA content,but their proline content increasing extents were among the top five.?4?Germplasms with clear drought-sensitive characteristics are the#30 from Inner Mongolia HeLanShan and the#7 from GanSu-TianZhu:being treated with drought,the increasing extents of the MDA content in the two were among the top two,but without significant increasing of proline content and activities of POD and SOD.?5?With the root from the Inner Mongolia TaoLi#8 and ABaGaQi#2,we did the transcriptome.We primarily analyzed the Ca²⁺binding protein-encoding genes and found SvCML1 with predicated Ca²⁺binding motif.However,with the purified Sv CML1 protein after being expressed exogenously,we demonstrated with Isothermal Titration Calorimeter technology that SvCML1 is not an Ca²⁺binding protein,instead of an protein specifically binding to Zn²⁺.?6?SvCML1 was found in the Endoplasmic Reticulum?ER?of the plant cells.Under relative high[Zn²⁺]growth condition,SvCML1 could increase the Zn content in the transgenic Arabidopsis roots and the Zn²⁺content in the ER.?7?Overly expressing SvCML1 rendered the transgenic Arabidopsis growth inhibition under relative high[Zn²⁺]and low[Fe²⁺]conditions.Lowering Fe²⁺significantly induced Zn accumulation Arabidopsis roots.Under the low[Fe²⁺]condition,SvCML1 transgenic Arabidopsis roots had higher amount of Zn than that in the wild type.Decreasing[Zn²⁺]in the medium could fully rescue the low[Fe²⁺]sensitive phenotype of the SvCML1 transgenic Arabidopsis.?8?SvCML1 could repress expression of Zn²⁺transporter genes At ZIF2?Zinc-Induced Facilitator2,At2g48020?and AtMTP1?Metal Tolerance Protein1,At2g46800?.Previous studies found that Arabidopsis lack of the two genes show growth inhibition phenotype under relative high Zn²⁺condition.?9?The Arabidopsis cDNA yeast library was screened with SvCML1 as bait with aim to identify SvCML1 interacting proteins.It led identification AtCLC2?Clathrin Light Chain2,At2g40060?.Arabidopsis lack of AtCLC2 or AtCLC3,another member of the AtCLC family,showed growth inhibition phenotype under low[Fe²⁺]condition.In summary,based on collection of germplasm and evaluation of growth as well as drought tolerance,we identified an transcript in the transcriptome of Sabina vulgaris encoding an calmodulin like protein.It was demonstrated with in vitro and in vivo experiments to be an Zn²⁺binding protein and could improve the plant's Zn content as well as the Zn dependent growth through multiple pathways.Results from this study provides theoretical instruction for Sabina vulgaris germplasm selection during being used in the ecological projects.Meanwhile,the study provides an functional gene for improving crops'Zn nutrition through molecular breeding.