Transcriptome of ZmPMP3g overexpressing maize inbred line Ye478, and wild type Ye478 under control, moderate drought, exogenous abscisic acid, or treatment combinations

This study is to identify the role of maize ZmPMP3g (GenBank accession no. EC869579.1), a proteolipid membrane potential modulator 3 (PMP3) domain gene, in drought tolerance of maize, and the genes and signaling pathways under over-expression of ZmPMP3g Overall design: The tillage soil from the experimental field of Guangxi University was collected, sun-dried, sieved, and then fully mixed with the local commercial organic fertilizer (8:2/w:w) as pot mix. The pot mix was loaded in pots (31 cm in diameter and 21 cm in height), 5 kg per pot. The pot mix of each pot was saturated with tap water and then monitored for moisture by using a Dong mei DT001 soil tester (Shanghai, China) equipped with a thin and long detector. When the moisture of the pot mix reached about 70%, pot experiment started. The maize ZmPMP3g (GenBank accession no. EC869579.1) ranging from start codon to stop codon was cloned into NcoI and BamHI restriction sites downstream of the 3×CaMV35S (3×200) promoter of plasmid pGSA1252 of Basta resistance gene, generating an expression construct, pGSA1252::ZmPMP3g. The construct was introduced into mature embryos of Chinese maize inbred line Ye478 by Agrobacterium tumefaciens strain LBA4404 mediated infection. The LBA4404-infected maize embryos were transferred onto Murashige and Skoog medium plates containing 0.1 mg/L IAA, 250 mg/L cefotaxime, 20 ppm herbicide Basta and 30 g/L sucrose, and then allowed to grow for 7 d at 28 °C under a cycle of light/16 h and dark/8 h. The regenerated Basta-resistant maize plantlets were transplanted into sandy soil in trays and then grew for 10 d at room temperature followed by foliar-spraying 40 ppm Basta once. The maize seedlings that survived 7 d after spraying Basta were transferred into pot mix and allowed to grow in the glass greenhouse at 20-25?°C and with 50%-60% air humidity under a cycle of light/16 h and dark/8 h. The transformed plants were subjected to identification by PCR, Southern blotting, and real-time quantitative PCR. Drought treatment started at 5-leaf stage of maize through natural mix drought without watering until to moderate drought (50% moisture for pot mix). The pot mix moisture in the parallel control was controlled at 70% by watering. When the pot mix was in moderate drought and severe drought respectively, sampling started and exogenous ABA was applied. The exogenous ABA application was conducted by spraying 15 mg/L ABA (Sigma, USA) on whole plants once while the leaves in control treatment were sprayed with an equal volume of tap water. The transcriptome sequencing was conducted on the second fully-expanded leaves down from the top of T4 transgenic line plants 9 d after spraying ABA on whole plants and parallel control plants, which were sampled at 10:00 a.m.

本研究旨在明确玉米ZmPMP3g（GenBank登录号：EC869579.1）——一种脂膜电位调节蛋白3（proteolipid membrane potential modulator 3, PMP3）结构域基因——在玉米抗旱性中的作用，以及过表达ZmPMP3g后相关基因与信号通路的变化。
实验设计：采集广西大学试验田耕作土壤，经晾晒、过筛后，与市售商品有机肥按8:2的质量比充分混合，作为盆栽基质。将基质装入直径31 cm、高21 cm的花盆中，每盆装5 kg。先用自来水将基质饱和，随后使用搭载细长探头的东美DT001土壤测试仪（中国上海）监测基质含水量，待含水量降至约70%时启动盆栽试验。
将玉米ZmPMP3g（GenBank登录号：EC869579.1）从起始密码子至终止密码子的序列克隆至携带草丁膦（Basta）抗性基因的pGSA1252质粒的3×花椰菜花叶病毒（Cauliflower Mosaic Virus, CaMV）35S（3×200）启动子下游的NcoI与BamHI限制性酶切位点之间，构建得到过表达载体pGSA1252::ZmPMP3g。采用根癌农杆菌（Agrobacterium tumefaciens）菌株LBA4404介导，将该载体转化至中国玉米自交系Ye478的成熟胚中。将感染农杆菌的玉米成熟胚接种于添加0.1 mg/L吲哚乙酸（IAA）、250 mg/L头孢噻肟、20 ppm除草剂草丁膦（Basta）及30 g/L蔗糖的Murashige和Skoog（MS）培养基平板上，于28 ℃、16 h光照/8 h黑暗的光周期条件下培养7 d。
将获得的抗草丁膦（Basta）再生幼苗移栽至托盘内的砂质土壤中，于室温下培养10 d后，叶面喷施一次40 ppm的草丁膦（Basta）。喷施草丁膦（Basta）后存活7 d的玉米幼苗移栽至盆栽基质中，置于温度20~25 ℃、空气相对湿度50%~60%、16 h光照/8 h黑暗光周期的人工气候温室中继续培养。通过聚合酶链式反应（PCR）、Southern印迹（Southern blotting）及实时定量PCR（real-time quantitative PCR）对转化植株进行分子鉴定。
于玉米五叶期启动自然干旱处理，停止浇水直至基质含水量分别达到中度干旱（50%）与重度干旱水平；平行对照组的基质含水量通过浇水维持在70%。当基质分别处于中度干旱与重度干旱阶段时，进行外源脱落酸（abscisic acid, ABA）喷施处理：处理组整株喷施15 mg/L的脱落酸（ABA，Sigma，美国），对照组喷施等体积的自来水。
于喷施ABA后9 d的上午10:00，分别采集T4代转基因株系及平行对照植株顶部向下第二片完全展开叶，进行转录组测序。