To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T0) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies.
We show that a vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E or Vegf27Cb have no effect, but simultaneous inactivation of all three Vegf genes phenocopied the receptor mutant, and ectopic expression of Vegf27Cb redirected migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and we speculate that the ancestral function of VEGF pathways was to guide blood cell movement. 相似文献
Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein. 相似文献
Few studies have examined the physiological/biochemical status of hepatocytes in patients with compensated and decompensated cirrhosis in situ. Phosphorus-31 magnetic resonance spectroscopy ((31)P MRS) is a noninvasive technique that permits direct assessments of tissue bioenergetics and phospholipid metabolism. Quantitative (31)P MRS was employed to document differences in the hepatic metabolite concentrations among patients with compensated and decompensated cirrhosis as well as healthy controls. All MRS examinations were performed on a 1.5-T General Electric Signa whole body scanner. The concentration of hepatic phosphorylated metabolites among patients with compensated cirrhosis (n = 7) was similar to that among healthy controls (n = 8). However, patients with decompensated cirrhosis (n = 6) had significantly lower levels of hepatic ATP compared with patients with compensated cirrhosis and healthy controls (P < 0.02 and P < 0.009, respectively) and a higher phosphomonoester/phosphodiester ratio than controls (P < 0.003). The results of this study indicate that metabolic disturbances in hepatic energy and phospholipid metabolism exist in patients with decompensated cirrhosis that are not present in patients with compensated cirrhosis or healthy controls. These findings provide new insights into the pathophysiology of hepatic decompensation. 相似文献
In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar. 相似文献
Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation. 相似文献
Vegetation changes are documented from a well-dated pollen record from Lake Emakat, Empakaai Crater, northern Tanzania. This pollen record includes the time interval covering the Pleistocene/Holocene transition, analysed at a resolution interval averaging 200 yr. Around the crater lake, an Hagenia-forest development starting at 14,500 cal yr BP lasted until 13,000 cal yr BP. A change in vegetation, indicated by an increased proportion of Nuxia congesta in the forest and Artemisia in the afro alpine grassland after 13,000 cal yr BP, corresponds in time to the Northern Hemisphere's Younger Dryas cooling. Grasses and sedges increased at 10,100 cal yr BP, indicating a significant increase in local pollen possibly attributed to lowered lake level, related to drier conditions. Although the Empakaai pollen record documents continuous forest conditions, from 14,500 to 10,100 cal yr BP, the variation in the proportion of forest components seem to respond to environmental changes at the millennium scale. 相似文献