马尾松花粉超低温保存的生理响应及转录组分析

张晓宁<sup>1</sup>; 张 烨<sup>1</sup>; 徐展武<sup>2</sup>; 覃子海<sup>1</sup>; 魏秋兰<sup>1</sup>; 林 东<sup>1</sup>; 肖玉菲<sup>1</sup>; 刘海龙<sup>1*</sup>

引用本文:	张晓宁, 张烨, 徐展武, 覃子海, 魏秋兰, 林东, 肖玉菲, 刘海龙.马尾松花粉超低温保存的生理响应及转录组分析[J].广西植物,2025,45(10):1916-1925.[点击复制]
	ZHANG Xiaoning, ZHANG Ye, XU Zhanwu, QIN Zihai, WEI Qiulan, LIN Dong, XIAO Yufei, LIU Hailong.Physiological response and transcriptome analysis to cryopreservation of masson pine(Pinus massoniana)pollen[J].Guihaia,2025,45(10):1916-1925.[点击复制]

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马尾松花粉超低温保存的生理响应及转录组分析
张晓宁¹, 张烨¹, 徐展武², 覃子海¹, 魏秋兰¹, 林东¹, 肖玉菲¹, 刘海龙^1*
1. 广西壮族自治区林业科学研究院/广西优良用材林资源培育重点实验室, 南宁530002;2. 湖北大学生命科学学院/生物催化与酶工程国家重点实验室, 武汉 430062

摘要:

为初步解析马尾松花粉对超低温保存的生理响应及相关代谢机制,该文以马尾松花粉为研究对象,在-196 ℃液氮进行超低温保存并分析冷存过程中[冻存前(CK)、液氮冻存后(LD)、化冻后(HD)]与活性氧(ROS)相关的生理指标及转录组数据。结果表明:(1)马尾松花粉超低温保存的最适含水量为3.96%,该含水量下将花粉直接投入液氮保存至少48 h,冻存前后花粉存活率分别为78.54%和73.80%。(2)超氧化物歧化酶(SOD),抗坏血酸过氧化物酶(GSH),谷胱甘肽(APX),抑制羟自由基能力4个指标在冻存前后差异显著。(3)转录组测序共获得65.60 Gb过滤数据,有38 505个基因比对到参考基因组(47.84%),CK vs LD、CK vs HD、LD vs HD的差异表达基因(DEGs数量分别为232、268、218个)。GO和KEGG分析表明,应激响应(response to stimulus)和抗氧化活性(antioxidant activity)等GO term显著富集; 植物激素信号转导、MAPK信号途径、淀粉和蔗糖代谢、果糖和甘露醇代谢、过氧化物酶体等KEGG途径显著富集,从这些途径中进一步筛选到10个可能与冷冻保存过程中损伤和修复有关的基因。该研究结果为马尾松种质资源高效利用和安全保存提供了技术手段,也为进一步解析冷冻保存损伤的分子机制提供参考。

关键词: 马尾松, 花粉, 超低温保存, 生理, 转录组

DOI：10.11931/guihaia.gxzw202403015

分类号:Q945

文章编号:1000-3142(2025)10-1916-10

基金项目:广西自然科学基金(2021GXNSFAA196069, 2016GXNSFBA380224); 广西优良用材林资源培育重点实验室项目(2020-A-03-01); 广西重点研发计划项目(桂科AB24010289)。

Physiological response and transcriptome analysis to cryopreservation of masson pine(Pinus massoniana)pollen

ZHANG Xiaoning¹, ZHANG Ye¹, XU Zhanwu², QIN Zihai¹, WEI Qiulan¹, LIN Dong¹, XIAO Yufei¹, LIU Hailong^1*

1. Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Guangxi Forestry Research Institute, Nanning 530002, China;2. State Key Laboratory of Biocatalysis and Enzyme Engineering, College of Life Sciences, Hubei University, Wuhan 430062, China

Abstract:

In order to preliminary analyze the physiological response and related metabolic mechanism of Pinus massoniana pollen to cryopreservation, the pollen of P. massoniana, as the research object, was cryopreserved in liquid nitrogen(LN), at -196 ℃. The physiological indexes related to reactive oxygen species(ROS)were assessed during the freezing process(CK: before freezing; LD: after freezing with liquid nitrogen; HD: after freezing and thawing). Transcriptome sequencing was also performed using the next generation sequencing( 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 directly immersed in liquid nitrogen, where it was stored for at least 48 h, then was removed from liquid nitrogen and transferred to room temperature for 20 minutes to achieve thawing. The survival rate was 73.80% after freezing and thawing, 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 from transcriptome sequencing, with 38 505 genes mapped to the reference genome(47.84%); the number of differentially expressed genes(DEGs)in comparisons of CK vs LD, CK vs HD, and LD vs HD were 232, 268, and 218, respectively. In the current study, Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses showed that GO terms such as response to stimulus and antioxidant activity; KEGG pathways such as plant hormone signal transduction, MAPK signaling pathway, starch and sucrose metabolism, fructose and mannose metabolism, and peroxisome pathways were significantly enriched. Ten genes in these pathways that may be closely related to protection and repair during cryopreservation were further screened. All the results indicate that the freezing and thawing process which is related to oxidative stress induced by ROS affects the cryopreservation of P. massoniana pollen, as well as the water content. The results of this study help to improve the efficient preservation and utilization of the pollen of Pinus massoniana, and also provide a reference for further analysis of the molecular mechanism of cryopreservation damage.

Key words: Pinus massoniana, pollen, cryopreservation, physiology, transcriptome