马尾松花粉超低温保存的生理响应及转录组分析

his study aimed to investigate the response mechanism of Pinus massoniana pollen tocryopreservation. Pollen of P. massoniana was cryopreserved within liquid nitrogen (LN), at-196 . Eleven physiological indexes related to reactive oxygen species (ROS) were assessedduring the freezing process (ck: before freezing, LN: after freezing, HD: after freezing andthawing). Transcriptome sequencing was also performed using the next generationsequencing( NGS) technology and Illumina HiSeq TM 6 000 platform. The results were as follows:(1) The optimal water content for cryopreservation was 3.96 % (fresh weight basis). At this water content, the P. massoniana pollen was transferred to 2 mL cryovials and directly immersed in liquidnitrogen, where it was stored for at least 48 h, then was removed from liquid nitrogen and transferred toroom tempreture for 20 minites to achieve thawing. The survival rate was 73.80 % after freezing andthawing, compared with 78.54 % before freezing. (2) During the cryopreservation process,significant differences were observed in the four indexes of superoxide dismutase (SOD),glutathione (GSH), ascorbate peroxidase (APX), and the ability to inhibit hydroxyl free radicals. (3)A total of 65.60 Gb clean data were obtained, with 38 505 genes mapped to the reference genome(47.84 %); The number of differentially expressed genes (DEGs) in comparisons between controland freezing treatment (CK vs LD), control and thawing treatment (CK vs HD), and freezing andthawing treatment (LD vs HD) were 232, 268, and 218, respectively. In the current study, GeneOntology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyseswere performed, and revealed a group of genes related to response to stimulus and antioxidantactivity; and genes encoded some key proteins in plant hormone signal transduction, MAPKsignaling pathway, Starch and sucrose metabolism, Fructose and mannose metabolism, andperoxisome pathways. Ten of DEGs screened in these pathways may be closely related toprotection and repair during freezing and thawing process. All the results indicate that the freezingand thawing process which related to oxidative stress induced by ROS affect the cryopreservationof P. massoniana pollen, as well as the water content. Our results help to improve the efficient ofpreservation and utilization of the pollen of Pinus massoniana, and also provided reference forfurther analysis of the molecular mechanism of its freezing mechanism.