| Rapeseed(Brassica napus L.)is the largest planted oilseed rape species in China,At the same time,extreme weather has occurred in recent years,and winter rapeseed is susceptible to the effects of the later spring cold,when it is in the stage of shoots,flowering and even podding periods,which has a serious impact on the yield and quality of rapeseed.The effects of low temperature during flowering period on the flower organs and seedlings of Brassica napus,and the response of related genes after low temperature stress are not clear enough.In this study,Brassica napus GZhui(strong cold resistance in winter)and 10B(weak cold resistance in winter)were used as materials,and were subjected to low temperature stress in an artificial climate room at the early flowering stage(day 12 °C/14 h,night 2 °C/10h).,set a total of 5 treatments1 d,2d,3d,4d and 5d: the normal growth environment(day 22°C/14h;night 18°C/10h)as the control,and carried out physiological and cytological studies on the vitality of leaves,flower buds,and pistils and stamens.At the same time,the cold tolerance genes were predicted by transcriptome sequencing combined with q RT-PCR,and the following research results were obtained:1.By measuring the changes of physiological indexes of moss stem leaves and rosette leaves under cold stress during flowering.The results showed that the changes of each physiological index of the leaves after treatment were more complicated.Superoxide dismutase(SOD)of treated samples was not significantly different to the control;peroxidase(POD)was more sensitive,and all treatments showed a significant increase,and the lower leaves were more elevated than the moss stem leaves,of which10 B was more elevated than GZ hui;catalase(CAT)was not significantly different between the two materials treated than the control;catalase activity of GZ hui moss leaves increased and then decreased with the increase of treatment,and rosette leaves showed a significant decrease compared with the control after treating three days.The rest of the treatments did not show significant differences,CAT activity of 10 B moss leaves was significantly higher than the control only after treating four days The CAT activities of rosette leaves were lower than the control.Malondialdehyde(MDA)content was significantly increased in both material treatments compared to the control,and 10 B was more elevated than GZ hui.The content of soluble sugar(SS),an osmoregulatory substance,changed significantly,with a significant increase in GZ hui and only a significant increase in 10 B at 4 d of treatment compared to the control.Soluble protein(SP)was not significantly different in both material treatments compared with the control.2.By measuring the pollen viability and stigma fertility of flowers that opened on the day of cold stress and buds that recovered after cold stress,it was found that low temperature during flowering did not affect the pollen viability of flowers that opened on the treatment day,while stigma fertility was significantly reduced.For the buds that suffered from cold stress and were transferred to normal environment to resume flowering,the buds larger than 3.0 mm were less affected by low temperature when treated for less than 4 d.The decrease in pollen viability and stigma fertility was not significant until treated for more than 4 d.For the buds smaller than 3.0 mm,the pollen viability and stigma fertility decreased gradually with the increase of treatment time(GZ hui The pollen viability and stigma fertility of buds smaller than 3.0 mm decreased gradually with increasing treatment time,and only 72.83% of pollen viability and low stigma fertility were observed after 3 d of treatment for 1.5 mm-2.0 mm buds.The earlier the developmental stage of the flower buds when they were subjected to low temperature stress,the more damage was done to their post-flowering androgynous organ viability.The smaller the buds were,the greater the damage.After 3d cold stress of GZ hui,the number of grains per pod by buds at P1 stage was 4.67,only 18.19% of the control,while the number of grains per pod by buds at P5 stage was 23.67,92.21%of the control.The number of seed per pod at P1 stage was 4.33 after 3 d of 10 B treatment,which was only 19.98% of the control;the number of seed per pod at P5 stage was 17.67,which was 81.54% of the control.By pollinating untreated stigmas with low-temperature treated pollens and untreated pollen with low-temperature treated stigmas,it was found that there was no difference in the number of seed per pod between the two.It indicated that the low temperature led to the unfruitful situation due to the decreased activity of pollen and stigma,rather than only one of them.3.The buds of GZ hui and 10 B smaller than 2.0 mm from the control,treatment2 d and treatment 3d.The results showed that there were 4286 differentially expressed genes between GZ hui control and treatment group,4541 differentially expressed genes between 10 B control and treatment group,and total of 1396 differentially expressed genes in the intersection of the two materials.By GO enrichment as well as KEGG enrichment of these 1396 differentially expressed genes revealed that most of them were annotated to photosynthesis,proline and arginine metabolism,circadian rhythm,linoleic acid metabolism and other pathways.The 1396 differentially expressed genes were further analyzed and divided into six groups according to their expression changes after treatment,among which the differential gene expression trends in Cluster1,Cluster2 and Cluster3 were the same in both materials.Using online databases to obtain the whole development periods expression maps of the above three groups genes,we finally selected Bna A08g10990 D is a gene annotated to the proline and arginine metabolic pathways,and Bna C08g38140 D is predicted to be co-expressed with ATHOOK DNA binding protein by online tools.Sequence analysis of Bna A08g10990 D after cloning revealed that it encodes 666 amino acids,with only one amino acid difference in the two materials,and no difference in protein sequence.The expression was lower in control of GZ hui and 10 B,and was significantly higher in both materials after GZ hui low temperature stress.This gene encodes a protein with a PLN02439 domain,related to spermidine decarboxylase and has an important role in polyamine synthesis,which is associated with plant resistance and reproductive organ activity.It indicated Bna A08g10990 D might paly a role in response to low temperature stress during flowering. |
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