Application of Salt Tolerance Unique Gene Sequence rt-st14173 of Changyehongsha in Plant Salt Tolerance Genetic Engineering
A long-leaf red sand, genetic engineering technology, applied in the field of plant biology, can solve problems such as the impact of subsequent analysis, and achieve the effects of low cost, large throughput and fast speed
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Embodiment 1
[0026] Embodiment 1. long-leaf red sand seedling cultivation
[0027] The seeds of Changye Hongsha were collected from the East Alxa-West Ordos area of Inner Mongolia Autonomous Region in September 2008. The plump seeds were selected and sterilized with 10% sodium hypochlorite for 15 minutes, and sterilized with ddH 2 O was washed three times and seeded in a 150ml Erlenmeyer flask containing 40ml MS medium. After 72 hours of dark culture, culture at 25°C, humidity 70%, 16h:8h light / dark conditions. The seedlings grow to about 10cm (about 15d), and are transferred to a large test tube with a height of 25cm and a diameter of 5cm for cultivation. The sterilized 1 / 2 Hoagland nutrient solution is changed every two days, about 50ml each time. Select three long-leaf red sand seedlings with similar growth as experimental materials. Before NaCl treatment, cut the stems and leaves of three seedlings appropriately; after treating the seedlings with 100mM NaCl, harvest appropriate amou...
Embodiment 2
[0028] Example 2. Preparation of RNA-seq cDNA library
[0029] a. Total RNA extraction
[0030] Use plant plus RNA regent (DP437, Tiangen, Beijing) to extract total RNA according to the instructions, digest the total RNA with DNase I (TaKaRa) for 45 minutes, take 1.5 μl for 1% agarose gel electrophoresis, and measure with Nanovue plus The 260 / 280 OD values were all between 1.9 and 2.1, and the 260 / 230 were all greater than 2.0. The control and salt stress treated samples were mixed in equal amounts to obtain two RNA pools: C21 and T43. Finally, Agilent2100 detection was performed on the two samples.
[0031] b. Preparation of sequencing library
[0032] The total RNA is enriched with Oligo (dT) magnetic beads, and then the mRNA is fragmented. Using the mRNA as a template, the first strand of cDNA is synthesized with six base random primers (random hexamers), and then buffer, dNTPs, and RNase are added. H and DNA polymerase I synthesize cDNA second strand. After the reve...
Embodiment 3
[0033] Example 3. Assembly of sequencing results and bioinformatics analysis
[0034] a. Assembly of sequencing results
[0035] Use the short reads assembly software SOAPdenovo (assembly software) to do de novo transcriptome assembly. SOAPdenovo first connects reads (reads) with a certain length of overlap (overlap) into longer fragments. These assembled fragments without N obtained through the reads overlap relationship are called Contig.
[0036] Comparing the reads back to Contig, the different Contigs from the same transcript and the distance between these Contigs can be determined through paired-end reads. SOAPdenovo connects these Contigs together, and the unknown sequence in the middle is represented by N, thus obtaining Scaffold. Further use paired-end reads to fill holes in the Scaffold, and finally obtain a sequence with the least N and no longer extension at both ends, which we call Unigene. If multiple samples of the same species are sequenced, the Unigene assem...
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