Method for improving Jatropha fruit yield by overexpressing jcarf19 gene
A technology for fruit yield and tung seeds, applied in horticultural methods, genetic engineering, plant genetic improvement, etc., can solve the problems of difficult operation, unsatisfactory effect, labor-intensive, time-consuming and high cost, and achieve good application prospects, rapid yield increase effect, and economical significant effect
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Embodiment 1
[0092] Example 1 Overexpression of JcARF19 gene increases Arabidopsis biomass and seed yield.
[0093] Step 1: Construction of expression vectors for transformed plants
[0094]First, use SEQ ID No.1 as a template to design amplification primers (see Table 1, JcARF19-Full length-F and JcARF19-Full length-R), and use the pCAMBIA1300 vector series (purchased from Beijing Tianenze Gene Technology Co., Ltd.) to transform pCAMBIA1300-2X35S vector (SEQ ID No.5, transformation method: introduce the sequence of 2X35S promoter-multiple cloning site-3HA tag-Nos terminator into pCAMBIA1300 backbone vector, see Jian Ye et al., 2014, Biotechnology for biofuels, 7:91), construct the binary expression vector pCAMBIA:35S:JcARF19 (JcARF19OE). Design amplification primers according to SEQ ID No.2 as template (see Table 1, JcIAA9-Full length-F and JcIAA9-Full length-R), utilize the transformed pCAMBIA1300-2X35S vector to construct binary expression vector pCAMBIA:35S:JcIAA9 (JcIAA9OE). Such a...
Embodiment 2
[0100] Example 2 Overexpression of JcARF19 gene increases Jatropha seed yield and size.
[0101] Compared with Embodiment 1, the present embodiment 2 has the same step 1, and the different steps 2 and 3 are as follows:
[0102] Step 2: Tissue culture, transformation and regeneration of Jatropha plants: use the cotyledons of Jatropha germinated in vitro (gifted by JOils, Singapore) as explants for the transformation and regeneration of Jatropha Jatropha, specific methods refer to (Qu Jing et al., 2012 , Biotechnology for biofuels).
[0103] Preparation of Agrobacterium for genetic transformation: Transform the Agrobacterium strain AGL1 with two plant binary expression vectors (i.e., JcARF19OE and JcIAA9OE, prepared in Example 1) respectively, using kanamycin (50mg / L) and Rifampicin (20mg / L) LB solid plate screening positive clones. Agrobacterium strains with positive clones were cultured overnight to reach OD 595 =0.7-1.0 spare.
[0104] Co-cultivation: Cut the cotyledons i...
Embodiment 3
[0114] Example 3 Overexpression of JcARF19 gene changes the expression of cell proliferation and cell volume-related genes in Jatropha seed endosperm cells.
[0115]The expression of cell proliferation and cell volume-related genes (see Table 1 for primers) in endosperm cells of Jatropha seeds overexpressing JcARF19 was detected. Each sample was 100mg, and each treatment had at least three biological replicates. Total RNA was extracted and genomic DNA was digested. After Nanodrop quantification, 2 μg of total RNA was used as a template, and cDNA was synthesized by MMLV reverse transcriptase. Take 2 μl cDNA as template, 0.4 μl 10 μm primer, 10 μl SYBR Green (TOYOBO), and then make up to 20 μl with ddH2O. The real-time PCR program was pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 40 cycles, and fill-in extension for 5 min. All samples were performed in three technical replicates. JcUbiquitin was used...
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