Studies On A Dwarf Locus BnDCL1 And A Plant Type Regulation Gene BnSPL7-1 In Brassica Napus L.

Key genes related to plant type traits have played very important roles in crop breeding in increasing lodging resistance and elevating the harvest indices of crop cultivars.Although there have been numerous works on dwarf plant type in Brassica napus,exploring new materials with compact plant types is still of significance in breeding,as well as in elucidating the underlying mechanisms.On the basis of previous works,Bn DCL1 conferring dwarf architecture was fine-mapped onto the chromosome C05,and analyzed by biotechnological approaches.In present research,function of short sequence gene Bn SPL7-1 partially characteristic of SPL7 subfamily has been explored to improve the plant type derived from Bn DCL1 in B.napus.The main results are summarized as follows:1.Studies on locus Bn DCL1（1）Phenotype of Bndcl1 muant:A novel dwarf mutant with down-curved leaf（Bndcl1）was isolated from an EMS-mutagenized B.napus line in our lab.At seedling stage,leaves of the Bndcl1 mutant have a sharply down-curved and crinkled phenotype,with short petioles,which contrasts with the wild-type leaves that are normal with long petioles.At the flowering stage,flower buds of Bndcl1 grow downward,while the F₁ and wild-type flower buds are normal.At the mature stage,plant height of the Bndcl1 mutant and F₁ was about 30 cm and75 cm,respectively,considerably shorter than that of the wild type（190 cm）.The dwarf plant type is compact.The mesophyll cells of mutant and wild-type were compared by paraffin section and toluidine-blue staining.The results showed that the mutant displayed defects in adaxial-abaxial patterning.The number of chloroplasts in mesophyll cells was observed by laser confocal microscopy,the results showed that the number of chloroplasts was significantly greater in Bndcl1,especially in the spongy mesophyll cells.Leaf photosynthetic property determination in Bndcl1 mutant demonstrated that photosynthesis efficiency and net photosynthesis rate were significantly increased in the Bndcl1 mutant,which suggests that photosynthetic performance was improved in the mutant.Antioxidant enzymes assay demonstrated that the activities of multiple antioxidant enzymes were increased while the ROS levels were decreased in Bndcl1 leaves,which might protect the photosystems of Bndcl1against photooxidative damage.The level of BR in the leaves of Bndcl1 was significantly lower than that of wild,the results showed that the BR-related pathway was abnormal in the mutants.BR sensitivity test results showed that Bndcl1 was insensitive to BR.（2）i TRAQ-based quantitative proteomic analysis:Mutant Bndcl1 and its wild type,were compared based on i TRAQ technique,and 943 differentially accumulated proteins（DAPs）were found.Plenty of DAPs related to BR pathway were identified.This can reasonably explain the Bndcl1-derived pleiotropic phenotypes,such as retarded growth,reduced plant height,shorter petiole,and down-curved leaf,that are exhibited in defective mutants in BR-related pathways.Functional enrichment analyses shown that the top enriched KEGG pathways of the up-accumulated proteins were“photosynthesis”.Moreover,proteins associated with oxidative stress response and photosystem II repairing were also up-accumulated in Bndcl1,which may help the mutant to maintain the photosynthetic efficiency under unfavorable conditions.Important DAPs associated with leaf polarity establishment were detected in Bndcl1,including ribosomal proteins,proteins involved in post-transcriptional gene silencing,and proteins related to brassinosteroid.Consistent with these findings,histological observation showed that the mutant displayed defects in adaxial-abaxial patterning.（3）Mapping of Bn DCL1:Our previous research had mapped the mutant locus.Present mapping with SNP and SSR markers used another population“Tapidor×Bndcl1”F₅,and has narrowed the mapping interval of Bn DCL1 to 102 kb.（4）Preliminary analysis of gene function:The mapping interval contain 4 genes as annotated in public databases,including Bna C05g30720D encoding CERK1,Bna C05g30730D encoding PP2A B’regulatory subunit,Bna C05g30740D encoding NTP3,and Bna C05g30750D encoding SPL7.By genomic DNA sequencing,it was found that among the 4 genes,1-bp mutation in Bna C05g30730D in mutant occurred,leading to early termination of its translated amino acid sequence,which may result in defects of BR signal transduction,while other 3 gene sequences do not differ between mutant and the wild type.This result together with the result from comparative quantitative proteomics analysis consistently indicated that PP2A B’gene mutation is responsible for the dwarf and compact phenotype in the mutant.Overexpression vector and RNA interference（RNAi）vectors for Bna C05g30730D were constructed.And these vectors,transformations into Bndcl1 and ZS11 were conducted with using an Agrobacterium tumefaciens-mediated transformation approach.Transformations into mutant Bndcl1 did not succeeded because the Bndcl1 mutant grows in a very small state,and usually died in the floral-dip transformation procedure due to invasion of disease.2.Studies on a plant type regulation gene Bn SPL7-1（1）Sequence analysis of Bn SPL7-1:Bna C05g30750D positioning in the mapping interval is 123 bp in length,encoding 40 amino acids.Bna C05g30750D has an incomplete SBP domain,and has a nuclear localization signal（NLS）.Bna C05g30750D can be regarded as the SBP family gene,named Bn SPL7-1 although it is extremely short.Because function of Bn SPL7-1 is unclear and we hope to improve the Bndcl1 plant type,transformation with this short gene was conducted as shown below.（2）Subcellular localization of Bn SPL7-1:Vector Bn SPL7-1::GFP fusion protein was constructed for subcellular localization.Tobacco leaves were transformed with Bn SPL7-1::GFP under the constitutive Ca MV35S promoter.Results indicated that Bn SPL7-1 is located in the nucleus,cell membrane and cytoplasm of tobacco epidermis cells.（3）Transformant expression:In order to investigate the biological function of Bn SPL7-1,over-expression vector was constructed.The transformation into Bndcl1succeeded fortunately.The T₂ generation transgenic lines exhibited normal flatten leaf and tall stature.This result indicated that this short sequence gene functions positively in regulation of plant height,the same as Os SPL7 functions in rice.Furthermore,the content of glucoside in leaves of transformants increased.This indicated that the properly overexpression brings about positive alteration of glucoside synthesis.Endogenous plant hormone determination indicated that the content of IAA reduced,the content of JA increased in transfromant leaves.（4）RNA-seq analysis:To explore the function of Bn SPL7-1,RNA-seq analysis was performed with the transformants overexpressing Bn SPL7-1.By RNA-seq,3539differentially expressed genes（DEGs）were detected.Among DEGS,numerous genes related to glucoside biosynthesis synthesis was up-regulated.This result agree well with the glucoside determination data.RNA-seq data also shown that glucoside biosynthesis synthesis was up-regulated and the auxin efflux carrier genes was down-regulated in transformant leaves,which causes the decrease of auxin polar transport.These results revealed that Bn SPL7-1 can play a positive regulation role in plant gowth although it contains part of SBP domain.