与灵芝孢子高产相关的功能基因的发掘

Ganoderma lucidum spores are the tiny basidiospores which originate from mature G. lucidum fruiting bodies. They are greatly favored by the vast number of consumers because they have such medicinal functions as anti-tumor, enhancing immuno-modulation, and so on. But their low yield results in their series products too expensive, which not only hinder them to promote human health, but also limits the development of the consumer markets of G. lucidum spore powder. Therefore, it is very important to breed some new varieties as quickly as possible that are high in the yield of G. lucidum spores. Hitherto there is no other reports of the varieties of high yield of G. lucidum spores but a variety of G. lucidum HuNongLingzhi No.1 used in this study. How can the varieties be bred as quickly as possible? Exploring the functional genes related to the yield of G. lucidum spores, which may significantly promote directive breeding of more varieties with high yield of G. lucidum spores by means of Marker-Assisted Selection in the near future..At present, the varieties of Ganoderma lucidum HuNongLingzhi No.1 and G. lucidum HuNongLingzhi No.4 have been acted as the parents, which G. lucidum HuNongLingzhi No.4 is very few in the yield of G. lucidum spores. And F1 generation strains have been obtained using protoplast hybridization between the above two varieties. The F1 generation strains have been cultivated and G. lucidum spores are collected. And then a great deal of monokaryon strains have been obtained, that are originated from those spores. .In this study, F2 segregation population will be obtained using the backcross between the above monokaryon strains and the two parents, respectively. And cultivation experiment was conducted to reduce the size of the F2 segregation population. And then they will be cultivated and G. lucidum fruiting bodies are sampled at three stages of the pileus starting formation, complete formation and beginning producing spores, respectively. And total RNA will be extracted. The candidate genes related to G. lucidum spores yield through the transcriptome sequencing analysis. The related functional genes will be further confirmed using the gene silencing technique. Some technical route and methods will be established in this project and have significant reference to other edible and medicinal fungi.

灵芝孢子是灵芝生长成熟期从菌盖弹射出的极其微小的担孢子，因具有抗肿瘤、增强免疫等药理功能而备受消费者青睐。但因其产量低导致其加工产品昂贵，而限制了它为人类健康造福，也阻碍了其消费市场的发展。因此要尽快培育出孢子高产的灵芝新品种。目前，除了本研究中使用的“沪农灵芝1号”外，国内外尚无其他灵芝孢子高产品种的报道。而找到与灵芝孢子高产相关的功能基因，就为今后利用分子标记辅助选择培育更多孢子高产新品种打下坚实基础。.本研究利用“沪农灵芝1号”和“沪农灵芝4号”（几乎不产孢子）的杂交F1代的后代单核菌株分别同两个亲本回交获得两个分离群，并进行栽培，在灵芝菌盖开始形成、完全形成及即将开始产生孢子等阶段取样，获得RNA样品，通过转录组分析确定同灵芝孢子高产相关的候选功能基因。利用基因沉默技术，进一步确定同灵芝孢子高产相关的功能基因。.本项目建立的技术路线及相关方法将会对其它食药用菌研究具有重要借鉴意义。

本研究利用“沪农灵芝1号”和“沪农灵芝4号”（孢子极低产）的杂交F1代的孢子单核菌株分别同两个亲本回交获得两个分离群A和B并进行栽培。以其灵芝孢子产量为指标，我们找到了其中孢子高产和极低产的回交后代HS和LS并进行栽培。在其原基、菌盖开始形成、菌盖形成并开始产生孢子、孢子喷射旺盛及孢子喷射结束等5个阶段取样，获得其相应的RNA样品，通过RNA-seq和生物信息学分析并参考已发表的研究报道初步选定同灵芝孢子高产相关的候选功能基因4个。通过荧光定量PCR试验验证，我们发现：1. 基因OZ_05322(该基因编码蛋白的部分序列同Schizosaccharomyces pombe的MSH3序列的相似性为61%)在回交后代LS的原基、菌盖形成并喷射孢子阶段是HS的2倍以上，在孢子喷射旺盛和喷射结束阶段也显著高于后者。有研究认为该基因的过表达会严重损害其碱基错配修复（mismatch repair，MMR）能力；2. 基因OZ_06595和OZ_02840在回交后代HS的孢子开始喷射、孢子旺盛阶段的表达量显著高于LS。基因OZ_06595编码蛋序列同Pleurotus pulmonanus的MSH4序列STPP1的相似性高达80%以上，有研究发现Pleurotus pulmonanus的stpp1基因与担孢子的形成有着密切的关系。而OZ_02840编码蛋白序列与Schizosaccharomyces pombe的减数分裂RNA结合蛋白1(meiotic RNA-binding protein 1）的相似性为30%，在S. pombe中，该蛋白对减数分裂非常重要；3.基因OZ_08219在HS和LS的4个阶段中的表达量的差异变化与孢子产量的表型并没有显著的对应关系。对孢子开始喷射、孢子喷射旺盛及喷射结束等三个阶段的灵芝子实体结构的电镜扫描结果与上述基因OZ_06595、OZ_02840和OZ_05322的表达差异存在稳定的相关性。因此我们初步推断，基因OZ_06595、OZ_02840和OZ_05322可能与灵芝担孢子的产生存在着密切的关系。